Protein translocation in Escherichia coli

ELSEVIER

Biochimica et Biophysica Acta 1197 (1994) 311-343

Biochim~ic~a et BiophysicaA~ta

Protein translocation in Escherichia coli Robert A. Arkowitz a, Martine Bassilana b,* a MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK b Laboratoire Jean Maetz, Commissariat ?t l'Energie Atomique, B.P. 68, 06230 ~llefranche-Sur-Mer, France Received 13 April 1994

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

312

Preprotein characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The leader sequence domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 The mature domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

314 314 315

Preproteins in the cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Preprotein folding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. General description of chaperonesy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. G r o E L / G r o E S and D n a K / D n a J . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. F f h / F f s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. SecB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. In vivo investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. SecB purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3. SecB preprotein binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4. SecB preprotein recognition and binding specificity . . . . . . . . . . . . . . . . . . . . . . 3.5.5. SecB-dependent and -independent preproteins . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.6. SecB preprotein targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

316 316 317 317 317 318 318 318 319 319 320 320

4. Preprotein translocation across the cytoplasmic membrane . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Translocation apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Genetic identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Temperature sensitive mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Biochemical identification of SecA and S e c Y / E . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. S e c Y / E / A organization and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5. SecA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6. SeeY and SecE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.7. Acidic phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Energetics of preprotein translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. ATP requirement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: IMV, inverted membrane vesicles; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PE, phosphatidyl ethanolamine; PG, phosphatidyl glycerol; PC, phosphatidyl choline; DOPC, dioleyl phosphatidyl choline; DOPG, dioleyl phosphatidyl glycerol; DOPE, dioleyl phosphatidyl ethanolamine; AMP-PNP, adenosine 5'-(/3,y-imino)monophosphate; D H F R , dihydrofolate reductase; BPTI, bovine pancreatic trypsin inhibitor; APDP, N-(4-(p-azido0304-4157/94/$26.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 3 0 4 - 4 1 5 7 ( 9 4 ) 0 0 0 1 1 - 5

321 321 321 322 322 323 324 325 327 328 329

salicylamido)butyl)-3'-(2'-pyridyldithio) propionamide; A/zH +, electrochemical proton gradient; A~, electrical gradient; ApH, chemical proton gradient; NMR, nuclear magnetic resonance, CD, circular dichroism, IR, infra red; LacZ, /3-galactosidase; ANS, 1-anilinonapthalene-8-sulfonate; TCA, trichloro-acetic acid; CL, cardiolipin, LPS, lipopolysaccharide. * Corresponding author. Fax: +33 93 766017.

312

R.A. Arkowitz, M. Bassilana / Biochimica et Biophysica Acta 1197 (1994) 311-343 4.2.2. A/xH + requirement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2.1. A/xH* in vivo and in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2.2. Possible roles of A#H + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. ATP/A/xH + interrelationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Other energy sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Environment of preprotein during translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . .

329 329 330 331 332 332

5. Subsequent steps of translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. SecD and SecF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Leader peptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Folding and disulfide bond formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Targeting and assembly into the outer membrane . . . . . . . . . . . . . . . . . . . . . . . . . . .

333 333 334 335 335

6. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

336

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

337

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

337

1. Introduction

of t h e s e o r g a n i s m s an d the ability to carry o ut g e n e t i c an d b i o c h e m i c a l investigations. T h e p r o t e i n c o m p o n e n t s involved in p r o k a r y o t i c s e c r e t i o n are well d e f i n e d in Escherichia coli ( r e f e r e d to as Sec-proteins), allowing i n d e p t h studies on t h e m e c h a n i s m o f p r e p r o t e i n translocation, e n e r g e t i c s of t r a n s l o c a t i o n , an d subseq u e n t steps following m e m b r a n e transit. T h e pr oc e s s o f p r e p r o t e i n t r a n s l o c a t i o n in E. coli r e q u i r e s n u m e r o u s p r o t e i n c o m p o n e n t s i n cl u d i n g soluble, p e r i p h e r a l a nd i n t e g r a l m e m b r a n e p r o t e i n s ( T a b l e 3). In addition, protein translocation requires energy from both A T P

T h e t r a n s p o r t of p r o t e i n s to d i f f e r e n t o r g a n e l l a r d e s t i n a t i o n s is an essential process. In e u k a r y o t i c cells, the t r a n s l o c a t i o n of p r e p r o t e i n s into t h e e n d o p l a s m i c reticulum, m i t o c h o n d r i a , a n d p e r o x i s o m e s results in the localization o f specific p r o t e i n s to th e i r r e s i d e n t c o m p a r t m e n t s . P r o t e i n s e c r e t i o n in p r o k a r y o t e s is m u c h m o r e restricted, with t r a n s l o c a t i o n o c c u r r i n g almost entirely across t h e cytoplasmic m e m b r a n e . This process has b e c o m e increasingly s t u d ie d d u e to th e simplicity

Table 1 Well-characterized preproteins Preprotein

Gene

Mr

Function

Resident Location

Chaperone

Ref.

37

porin?

OM

SecB

59,100,361

a

Outer membrane protein A proOmpA ompA Outer membrane protein F proOmpF ompF Maltose binding protein preMBP malE

39

porin

OM

SecB

100,121,362

43

maltose binding protein

P

SecB

59,121,126,363

Ribose binding protein preRBP rbsB

31

ribose binding protein

P

Ffh

100, 113,144, 364

50

maltose transport

OM

SecB

121,122,365

42 32

antibiotic resistance

P

GroEL/ES, Ffh

100, 113

8

stabilize OM

OM

(not SecB)

360,366

49

phosphatase

P

DnaK/J, Ffh

100, 121,367, 368

39

porin

OM

SecB

124, 369

Lambda phage receptor preLamB lamB /3-Lactamase preBla-Amp C bla preBla-TEM Lipoprotein preLpp lpp Alkaline phosphatase prePhoA phoA Inorganic phosphate porin prePhoE phoE

a OM, outer membrane; P, periplasmic.

R.A. Arkowitz, M. Bassilana / Biochimica et Biophysica Acta 1197 (1994)311-343

313

Table 2 Artificial preproteins Fusion proteins

ProOmpA covalent derivative b

Preprotein

protein fused to a

Ref.

preMBP preLamB prePhoE prePhoA proOmpA proOmpF prePhoA preBla preBla proOmpA proOmpF

LacZ LacZ LacZ LacZ LacZ LacZ Biotination box Biotination box TIM DHFR Lpp

41, 154, 370 4, 371 373 372 76 374 390 390 45, 49 76, 78 253, 360

Ref. proOmpA-BPTI

77, 176

proOmpA-Lpp

273

proOmpA-peptide

273

proOmpA-disulfide loop

77, 270, 272

proOmpA-coumarin proOmpA-APDP proOmpA-NEM

82 211 77

a TIM, triosephosphate isomerase; DHFR, dihydrofolate reductase. b BPTI, bovine pancreatic trypsin inhibitor; APDP, N-(4-(p-azidosalicylamido)butyl)-3'-(2'-pyridyldithio)propionamide; NEM, N-ethylmaleimide.

hydrolysis and the proton electrochemical gradient (A/xH +) to drive the unidirectional transport of preproteins across the membrane. Protein secretion across the bacterial cytoplasmic membrane is comprised of different steps. Preproteins destined for secretion are targeted to the inner membrane and maintained in translocation competent states by cytosolic chaperones. Subsequently, translocation across the cytoplasmic membrane occurs, with the preprotein maintained in a largely unfolded state. Upon translocation across the membrane, preproteins are processed to mature proteins, folded to their active conformation, and in some cases assembled into higher order structures. This entire process is refered to as protein secretion. This review will examine the general process of protein secretion (sec-dependent translocation) used by a number of periplasmic and outer mem-

LEADER

~5

~'~

MATURE DOMAIN

SEQUENCE

++

brane proteins and will not discuss specific protein transport systems such as haemolysin [1,2]. Protein secretion research can be characterized by two different approaches: in vivo genetic studies and in vitro biochemical studies. The combination of these two approaches has enabled the investigation of such a physiological process at a biochemical level. Genetic studies have provided a vast amount of information on the biological function of proteins involved in secretion. On the other hand, the biochemical studies have allowed the molecular characterization of translocation components and their interactions. Both in vivo and in vitro studies of protein secretion have taken advantage of physiological and model substrates (Tables 1 and 2). This review will focus on sec-dependent protein secretion in the gram-negative bacteria E. coli. Certainly the well developed genetic tools in E. coli have

y -3

-2

-

ala gly thr val

x

ala gly ser

0

Fig. 1. Schematic representation of a typical preprotein. Positively charged, hydrophobic, and hydrophilic regions of the leader sequence are illustrated. The amino acid residues required for leader peptidase cleavage at the leader-mature junction are shown. In addition, a helix breaker residue is required at position - 6 to - 4 .

314

R.A. Arkowitz, M. Bassilana / Biochimica et Biophysica Acta 1197 (1994) 311-343

made this organism ideal for secretion studies. As a result many preproteins and protein components involved in protein translocation from E. coli have been overexpressed, purified to homogeneity, and biochemically characterized.

2. Preprotein characteristics Proteins that are destined for translocation across an intracellular membrane are termed preproteins which have two domains, the amino-terminal leader domain and the mature domain. These precursors are typically synthesized with amino-terminal extensions called leader or signal sequences ranging from 15 to 30 amino acids in length [3]. Table 1 shows a representative sample of well characterized E. coli preproteins along with their molecular weight, cellular function, location and required chaperones. Interestingly, there are no sequence identities between preprotein leader sequences, yet the positively charged and hydrophobic nature of this domain is a conserved feature throughout preproteins. While the leader sequence is essential for efficient translocation, determinants in the mature domain of preproteins also contribute to translocation efficiency [4,5]. There have been a large number of studies on wild-type (Table 1) and artificial preproteins (Table 2), examining their folding, translocation competence, interactions with lipid bilayers, translocation in vitro, and in vivo. This section will concentrate on the common features of preprotein leader and mature domains and their roles in translocation. 2.1. The leader sequence domain

Analysis of many leader sequences has revealed that this domain is comprised of an amino-terminal region consisting of 1-3 positively charged residues, a central region consisting of 10-15 hydrophobic residues, and a hydrophilic carboxy-terminal region (for review see [6,7]) (Fig. 1). The positively charged residues in the amino-terminal region are not essential for leader sequence function in vivo [8-12], yet appear to be necessary for translocation in vitro [11,392]. The subsequent hydrophobic stretch is necessary for secretion [13-19]. The conservation of these two regions in leader sequences suggests that leader sequences interact both electrostatically and hydrophobically with the cytoplasmic membrane. The hydrophilic carboxy-terminal region of the leader sequence is involved in leader peptidase recognition and cleavage (discussed later). A number of different biophysical techniques have been used to demonstrate that leader peptides interact with model membrane monolayers and bilayers. The propensity of leader peptides to interact with mere-

branes correlates well with the ability of the respective preproteins to translocate in vivo [20-22]. Leader peptides of preLamB [20,21,23] and prePhoE [24,25] bind and insert into phospholipid monolayers. This interaction requires negatively charged phospholipids and the hydrophobic central core of the leader domain [24,26]. Using fluorescence and NMR spectroscopy, it was demonstrated that leader peptides of prePhoA [27], p r e L a m B [20,28,29], prePhoE [24,25,30,31] and proOmpA [22] significantly perturb the lipid bilayer structure, resulting in lipid vesicle aggregation and increased permeability. Bilayer resident quenchers such as nitroxide spin labelled [29] or brominated lipids [30], have been used to assess the bilayer insertion of preLamb or prePhoE leader peptides, respectively. The hydrophobic central core of both of these leader peptides penetrates deeply into the bilayer. These studies all indicate that a central property of leader sequences is their ability to interact with and insert into lipid bilayers. Biophysical and genetic studies on leader sequence domains demonstrated that their ability to adopt an a-helical conformation in a hydrophobic environment [32] correlates with biological activity [21,22,28,33]. Leader peptides in an aqueous environment appear to be unstructured [22,28,32] and adopt a /3-structure upon interaction with a lipid monolayer surface [23,32]. Insertion of leader peptides into a lipid monolayer results in a preference or stabilization of an a-helical conformation [22,23,32,33]. It has been suggested that a conformational change in the leader domain from a /J-structure to an a-helix within the lipid phase may drive the insertion of 10-15 residues of the mature domain [24,32]. Circular dichroism and fluorescence spectroscopy show that a LamB leader peptide inserts into lipid vesicles concomitant with a conformational transition from random coil to a-helical structure [34]. NMR spectroscopic analyses indicate that the a-helical structure begins at the leader peptide hydrophobic core with its amino-terminus interacting with the membrane surface [34]. These studies all support the notion that leader sequence domains undergo major conformational changes upon movement from a hydrophilic to a hydrophobic environment. This conformational flexibility appears to be an intrinsic property of leader sequences. The positively charged amino-terminus and the hydrophobic core are important for the conformational flexibility, ability to insert into model membranes, and in vivo function of leader sequences. A direct interaction of a preprotein leader sequence domain with any component (lipid or protein) of the cytoplasmic membrane has not been demonstrated during preprotein translocation. Nonetheless, recent studies show a correlation between the phosphatidylglycerol requirement for preprotein translocation (see below) and the re-

R.A. Arkowitz, M. Bassilana /Biochimica et Biophysica Acta 1197 (1994) 311-343

quirement for positive charges at the amino-terminus of the preprotein leader sequence [35]. Although the isolated leader peptides of different preproteins have been extensively studied, less is known about the interactions between the leader and mature portion of preproteins. Biophysical studies have shown that the amino-terminal 28 residues of the mature domain of preLamB have no effect on the conformation or membrane binding properties of the leader sequence [29]. Conversely, the leader sequence of preRBP and preMBP retard the folding of the mature portion of their respective preprotein [36]. Functional interactions between the leader sequence of RBP [37] or MBP [38] and their mature domains also have been suggested by the isolation of intragenic suppressors of leader sequence mutations. Mutations in the mature domain of these preproteins were isolated which slowed folding and therefore increased the time in which these preproteins were competent for translocation [37,38]. The role of preprotein folding in translocation will be discussed in more detail in section 3. These structural studies show that leader sequences have distinct physical properties which are important for their function in translocation.

2.2. The mature domain This section will discuss the role of the mature domain in preprotein translocation with emphasis on the size of the preprotein and the ability of a variety of artificial preproteins to translocate across the cytoplasmic membrane. It is difficult to generalize with respect to the mature domain, due to the complete lack of sequence similarity in preprotein mature domains. Many different secretory proteins (Table 1) use a common translocation pathway, suggesting the absence of a common primary sequence requirement within the mature domain. Nevertheless, this lack of common primary sequence does not rule out a conserved structural element such as that observed in leader sequences. Attachment of a leader sequence to most cytosolic proteins (but not all, see [39]) does not result in their translocation [4,40-43], strongly indicating that some feature of the preprotein mature region is necessary for translocation. Numerous studies have shown that the mature portion of the preprotein contributes to the efficiency of export (for review see [44]). For example, at least 28 residues of the amino-terminus of mature LamB (in addition to the leader sequence) are necessary for the secretion of a LacZ fusion protein [5] and more than 8 residues of/3-1actamase are necessary for efficient secretion of a triosephosphate isomerase chimera [45]. In addition, mutations near the aminoterminus of the mature domain [43,46], particularly the insertion of charged residues, inhibit the translocation

315

of different preproteins (for example see [47-51]). The cause of this inhibition could be due to multiple effects such as deleterious interactions of these positive charges with acidic phopholipids, leader peptidase, SecA, or the effect of these charges on preprotein structure or translocation energetics. Although much effort has been focused on the effect of hydrophobicity, charge, and size of the preprotein mature domain on its translocation (for review see [44]), the conclusions from such studies must be taken cautiously, because of the possibility of indirect effects from such alterations. The insertion of long hydrophobic stretches (longer than 15 amino acids) into the mature portion of proOmpA [52] or preLpp [53] blocks translocation of these outer membrane proteins resulting in their attachment to the cytoplasmic membrane, whereas the insertion of shorter or less hydrophobic sequences into the proOmpA mature domain did not inhibit its translocation. Preproteins with varying numbers of charged residues in their mature domains (with the exception of amino-terminus) can translocate efficiently. At the extreme, proOmpF-Lpp can translocate efficiently even in the absence of charged residues in its mature portion [54], suggesting that charged residues in the mature domain are not required for translocation. Similarly, deletions of portions of the mature domain of preproteins have shown that preprotein transiocation is quite tolerant to such drastic alterations [5,55-63]. For example, deletion of residues 1-229 of the 325 residues proOmpA has no effect on its localization in vivo [62,63]. In vivo studies with truncated derivatives of proOmpA have indicated that there exists a lower limit in the size of preproteins translocated across the cytoplasmic membrane [63]. A proOmpA derivative with 67 amino acid residues in the mature domain is localized to the periplasm, whereas a derivative with 51 amino acids in the mature domain remains unprocessed in the cytosol. In contrast, a proOmpF-Lpp fragment containing only 45 amino acids in the mature domain [54] and a proOmpA fragment containing 53 amino acids in the mature domain [64] translocate in a sec-dependent fashion in vitro. This discrepancy between in vivo and in vitro results is likely attributed to the greater complexity of secretion in vivo since translocation in vivo requires selectivity (due to the myriad of other preproteins) and preprotein stability. In addition, there may be different rate limiting steps between in vivo and in vitro translocation. If there is a minimum size of the mature domain required for interaction with Sec-proteins, one would predict that there might be a size limit for sec-dependent translocation in vitro. Identification of this limit size must await further studies. No unique primary sequence within the mature domain is required for translocation, yet the mature do-

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main is important for translocation. Comparison of the translocation of sec-independent M13 procoat and secdependent proOmpA by examining chimeras of these two preproteins suggests that sec-dependence is dictated, in part, by the mature domain [65]. In addition, the folded state of the mature domain appears to be important for translocation [66,67] (further discussed in the 'Role of chaperones' section). More recently, it has been demonstrated that small amounts (1-2% of the total) of both PhoA and Bla lacking leader sequences can translocate across the cytoplasmic membrane [68,69]. Suprisingly, signal sequence suppressor mutations in SecY can increase the efficiency of export of leaderless PhoA which appears to be secreted in a sec-dependent fashion [69]. These studies suggest that leader sequences are not absolutely essential for translocation but rather that properties intrinsic to the mature domain, such as its ability to fold slowly, may facilitate preprotein translocation. Table 2 shows a representative list of fusion and covalent derivatives of preproteins which all translocate across the cytoplasmic membrane under defined conditions, demonstrating the nonspecific nature of the translocation apparatus. In vivo studies have taken advantage of the translocation characteristics of the fusion proteins to develop genetic screens for translocation c o m p o n e n t s . Covalent derivatization of proOmpA has enabled an in depth examination of the ability of the translocation apparatus to translocate some secondary and tertiary structural elements. These studies have focused on the energetic requirements for translocation (discussed in section 4.2). In addition, covalent modification has made it possible to attach specific probes at unique locations in the preprotein. This section has emphasized the characteristics of the leader sequence and the mature domain of preproreins. It is increasingly clear that a preprotein is not just any protein with a leader sequence attached to its amino-terminus. Leader sequences, which have a number of conserved physical characteristics and mature domains, although less well-defined, both play a significant role in preprotein translocation. In thinking about preproteins, we must be careful not to oversimplify our analysis by restricting it to primary sequence comparisons. Both the secondary and tertiary structure, and equally important, the lack of structure (or ability to fold slowly) of preproteins, appear to be important in the translocation process. Indeed, it has been suggested [70] that interactions between the leader and mature domain may result in a structure or state of the preprotein that is essential for translocation. The process of export across the cytoplasmic membrane is comprised of events taking place in the cytosol, such as protein synthesis, interactions with chaperones, targeting to the cytoplasmic membrane, and translocation across this membrane. The next chapter will examine

the state of preproteins in the cytoplasm and discuss their interactions with cellular chaperones.

3. Preproteins in the cytoplasm Preprotein translocation in E. coli occurs largely post-translationally. For example, preMBP, proOmpA, prePhoA, and prearabinose binding protein initiate translocation after reaching 80% or greater of their full length whereas preRBP, preBla, and preLamB are processed entirely post-translationally [71-73]. As a result, preproteins reside in the cytoplasm subsequent to synthesis and prior to translocation across the cytoplasmic membrane. Preproteins are typically exported with half-lives of 3-12 seconds [73]. Preproteins in the cytosol must be maintained in a loosely folded state in order to be translocation competent. In addition, nonproductive homo- and hetero-interactions, such as aggregation and nonspecific binding must also be prevented. Cellular chaperones such as G r o E L / G r o E S , D n a K / D n a J , and SecB interact with preproteins maintaining translocation competence. These chaperones may also be involved in targeting preproteins to the translocation sites on the cytoplasmic membrane. Targeting can be thought of in a thermodynamic sense, that is a final yield, a n d / o r kinetically, in which the rate of targeting is crucial such that competing nonproductive reactions like aggregation and folding do not persevere [66].

3.1. Preprotein folding Preproteins must be 'loosely' folded and not stably, 'tightly' folded in order to be translocated. PreMBP is no longer competent for translocation after folding into a compact proteinase-resistant conformation [66]. A number of in vivo and in vitro studies have confirmed that large stably-folded structures can block translocation by occupying translocation sites. Preproteins fused to LacZ [74-76] and D H F R [76] block secretion when expressed at high levels. In vitro, the covalently-folded BPTI [77] and the stably folded D H F R ternary complex [78] both block translocation in chimeric preproteins, forming transmembrane intermediates. Both the leader and mature domains of preproteins play a role in preventing or retarding the final proteinase resistant conformation that is characteristic of periplasmic proteins. An attached leader sequence retards the folding rates of the periplasmic proteins MBP [36,79], RBP [36,37], and Bla [80] 34-fold, 2-fold, and 15-fold, respectively. In the absence of a leader sequence, these periplasmic proteins are proteinase resistant [36,81]. Precursors of the outer membrane proteins OmpA [67,82] and PhoE [83,84] aggregate and

R.A. Arkowitz, M. Bassilana / Biochimica et Biophysica Acta 1197 (1994) 311-343

rapidly become incompetent for translocation after dilution out of denaturant, making it difficult to assess the role of the leader sequence in folding. The role of the mature domain in modulation of the preprotein folded state has been examined by isolation of intragenic suppressors of preproteins showing reduced export due to leader sequence mutations. Point mutations in both preMBP [79,85] and preRBP [37] mature domains have been isolated which restore translocation of these leader sequence mutant preproteins. These mutations in the mature domain all decrease the stability of the folded state of preMBP and preRBP and in addition decrease their rates of folding [37,38,79]. The similar effects of these mutations on export in vivo and folding in vitro suggest that the same rate limiting step determines folding in both cases [79].

3.2. General description of chaperones Chaperones are proteins which 'mediate the correct assembly of other polypeptides' [86] thereby 'modulating protein conformation' [87]. The reader is directed elsewhere for comprehensive reviews on chaperones [86-88]. A primary role of chaperones is facilitating protein folding under physiological and stress conditions by binding unstable states of proteins. Four cellular chaperones in E. coli have been shown to play a role in protein secretion: D n a K / D n a J , G r o E L / GroES, Ffh/Ffs, and SecB. SecB is the primary chaperone of the secretion pathway (see Table 1), most likely because of its specific interactions with the other Sec-proteins.

3.3. GroEL / GroES and DnaK / DnaJ G r o E L / G r o E S is a well characterized chaperone which exists as an oligomeric structure composed of a 14-mer of GroEL, as a double heptameric toroid, and a 7-mer of GroES as a single heptameric toroid bound at one end of the GroEL cylinder [89-93]. The first indication that GroEL was involved in the preprotein secretion came from crosslinking studies which demonstrated that newly synthesized preBla forms a complex with GroEL and that this complex stabilizes preBla for post-translational translocation [94]. Subsequently, similar crosslinking experiments showed that preBla not only interacts with GroEL, but also with GroES in this complex [95]. In vitro studies demonstrated that G r o E L / G r o E S can also stabilize proOmpA for posttranslational translocation by forming a soluble, stable 1:1 (molar ratio) complex [96]. In addition, G r o E L / GroES was shown to facilitate the in vitro folding of preBla in an ATP-dependent fashion [97]. This facilitation of folding is only apparent with preBla and not Bla, most likely due to the different rates of folding of these two species. These studies establish biochemi-

317

cally a functional interaction between G r o E L / G r o E S and preproteins. Consistent with the implicated role of G r o E L / GroES in protein secretion, overexpression of these proteins can efficiently suppress secA and secY ts phenotypes [98]. More directly, overexpression of either G r o E L / G r o E S or DnaK can facilitate the secretion of the poorly exported preLamB-LacZ fusion protein [99]. These genetic studies suggest a role of G r o E L / G r o E S and DnaK in protein secretion. To examine the specificity of G r o E L / G r o E S function in protein secretion, the effect of ts groEL mutations on kinetics of preprotein secretion was investigated [100]. In vivo translocation of preBla is significantly affected by mutations in GroEL/GroES, whereas preMBP, prePhoA, proOmpA, proOmpF, or preLpp translocation is minimally affected, demonstrating the specificity of the requirement for G r o E L / G r o E S in preprotein secretion. A role for D n a K / D n a J in preprotein secretion has been demonstrated using dominant dnaK and dnaJ mutations which strongly affected prePhoA secretion [101]. Consistent with the redundant function of cellular chaperones in preprotein secretion, a secB null strain is dependent upon D n a K / D n a J for preLamB and preMBP secretion. Conversely, D n a K / D n a J overexpression can partially substitute for SecB function in a null strain [101]. These experiments indicate that D n a K / D n a J are involved in preprotein secretion, yet direct biochemical evidence is necessary to support this contention. Both G r o E L / G r o E S and D n a K / D n a J are ATPases which are stimulated by unfolded proteins [89,91,94,96,102-104]. Structural studies have indicated that DnaK binds polypeptides in an extended conformation [105], whereas GroEL binds amphipathic ahelices [105,106]. Protein folding by these chaperones appears to occur in a stepwise fashion, via ATP-dependent release of the protein from the chaperone [107]. Recent experiments examining in vitro folding reactions in the presence of chaperones, suggest that DnaK, DnaJ, and G r o E L / G r o E S act sequentially with D n a K / D n a J stabilized polypeptides (prevented from misfolding and aggregation) serving as substrates for GroEL/GroES, which ultimately mediate correct folding [93]. Consistent with this order of events along a folding pathway, nascent ribosome-bound polypeptide chains have been shown to interact with DnaJ [108].

3.4. Ffh / Ffs Ffh and ffs encode E. coil homologs of the mammalian 54 kDa subunit of signal recognition particle (SRP54) and 7S RNA of SRP, respectively [109-111]. Both of these genes are essential for cell viability [112,113]. In vivo and in vitro, Ffh and Ffs are associ-

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ated, forming a ribonucleoprotein complex similar to their mammalian counterparts [114,115]. In addition, mammalian 7S R N A can functionally replace E. coli ffs in vivo [114], whereas Ffh can be reconstituted into mammalian SRP replacing SRP54 and allowing specific binding to signal sequences [116]. Cross-linking experiments [117] have demonstrated that the F f h / F f s ribonucleoprotein complex functions as signal recognition particle by specifically interacting with the signal sequence of a nascent secretory protein such as preprolactin and rnurein lipoprotein. Despite the large amount of information on the structure and interactions of F f h / F f s , the function of this ribonucleoprotein signal recognition particle is less well understood. Two different approaches have been used to examine the function of Ffs in vivo, regulated depletion [114] and induction of a dominant lethal mutation [115]. Loss of Ffs function by either method does not result in a secretion defect with proOmpA, preMBP, prePhoA, or preRBP, whereas secretion of preBla is substantially affected. It is difficult to assess the functional significance of the effect on preBla secretion, because both depletion of Ffs and induction of the dominant lethal mutant result in a heat shock response [94,100]. In vivo depletion of Ffh revealed a significant kinetic secretion defect for preLamB, preMBP, preRBP, prePhoA, and proOmpF [113] and a complete block of preBla secretion. PreBla, preRBP, and prePhoA, all SecB-independent preproteins, show the largest defect in secretion, leading to the suggestion that F f h / F f s functions as a preprotein-specific chaperone. Definitive interpretation of these secretion defects will require the demonstration that Ffh depletion does not result in a heat shock response, as does Ffs depletion. Further biochemical studies will be needed to address the function of F f h / F f s . 3.5. SecB

SecB is the most well characterized Sec-protein. Not only was it the first identified, but it is a small, soluble, and inherently stable protein (for recent reviews see [118,119]). Mutations in secB were isolated by their ability to prevent the entrance of preMBP-LacZ into the secretion pathway [120]. Mutations in secB result in pleiotropic protein secretion defects, with the severity of the defect varying with preprotein [120,121]. Mutations in secB confer both a kinetic defect in secretion and a decrease in yield of exported proteins in vivo [59,121-124]. Deletion of secB revealed that it is a nonessential gene, although null strains do not grow on rich media plates [121]. A heat shock response, yet notably not overexpression of DnaK alone or G r o E L / G r o E S , can substitute for secB function, suggesting that a basal level of unidentified heat-shock proteins is necessary for a secB null strain to grow

[125]. These results suggest that heat shock proteins and secB are functionally redundant. 3.5.1. In vivo investigations

In vivo protein secretion studies provided much information on the function of secB. In particular, studies on the secretion of preMBP mutants indicate different dependencies upon secB function. Wild type preMBP secretion is only reduced in the absence of SecB, whereas a mutation in its leader sequence renders preMBP secretion completely dependent upon secB [59,126]. Intragenic suppressor mutations in preMBP leader sequence were isolated which increased its secretion compared to wild type preMBP in a secB-background [126]. Strikingly, a double mutant carrying these suppressor mutations and a mutation in the preMBP mature domain, which slows folding, now translocate in a s e c B - i n d e p e n d e n t fashion [126]. These observations led to the suggestion that SecB affects preprotein folding. Consistent with this hypothesis, only in a secB null strain when export is blocked by the uncoupler CCCP, does a portion of preMBP fold into a proteinase resistant conformation [127]. Similarly, during in vitro synthesis preMBP becomes proteinase resistant in the absence of secB [59]. Another in vivo approach which implicated a function for SecB early in the protein secretion pathway was the study of export-defective preproteins. The expression of export-defective preproteins such as mutant preLamB or preMBP interfere with the secretion of wild-type preproteins [128] suggesting that the mutant preproteins are recognized at an early stage in the secretion process and compete for a limiting component. This limiting component was subsequently identified as SecB [59], providing strong evidence for the in vivo association of SecB with preproteins. The region of mutant preMBP [59] and preLamB [122] necessary to elicit an interference of preprotein secretion was mapped to residues 151-186 and 320-380 (regions well into the mature domain), respectively. In preLamB, the region residue 320-380 is not required for secretion of the wild type preprotein, yet it is required in the presence of leader sequence mutations [123]. These results suggest, as will be discussed in greater detail subsequently, that the leader sequence and a region in the mature domain of the preprotein are both necessary for SecB interactions. 3.5.2. SecB purification

The definitive demonstration of the role of SecB in modulation of preprotein folding came with the purification of this protein. SecB is a soluble oligomeric protein composed of 17 kDa subunits [129-131]. This purified protein enhances in vitro translocation of preMBP, quantitatively retards the folding of this pre-

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protein in the absence of membrane vesicles, and greatly increases the time in which preMBP is competent for translocation [129]. Using a biochemical approach, a cytosolic export factor was purified [131], shown subsequently to be identical to the secB gene product [132]. This protein is necessary for post-translational translocation of a mutant preMBP synthesized in a wheat germ translation system, and greatly stimulates (approximately 8-fold) the in vitro translocation of preLamB [133]. Similarly, purified SecB substantially stimulates (about 6-fold) prePhoE [83] and p r o O m p A [130] in vitro translocation and maintains both prePhoE and proOmpA in a translocation-competent state upon dilution of these preproteins out of denaturant [83,96]. The congruence between the in vivo genetic studies and the biochemical function of SecB firmly establishes a role of this protein in secretion.

3.5.3. SecB preprotein binding The purification of SecB has allowed biochemical characterization of its interactions with preproteins. Initially, in vitro translation reactions were fractionated by density gradient centrifugation in order to demonstrate co-sedimentation of SecB and preprotein [133]. In addition, immunoprecipitation techniques have been used to show specific binding between preproteins and SecB in both cell lysates (in vivo) [123,134,135] and crude in vitro translation systems [124,136,137]. In vivo studies indicate that SecB binds selectively and transiently to nascent preMBP, preLamB, proOmpF, and proOmpA but not prePhoA [134,135]. Using in vitro translation reactions, efficient co-immunoprecipitation of preMBP with anti-SecB sera was shown to depend upon the folding properties of the preprotein [136]. Specifically, preMBP deletion mutants unable to form wild-type tertiary structure and a point mutant that decreases the rate of folding [38,81], are co-immunoprecipitated [136]. These results suggest that SecB binds unfolded preproteins or partially unfolded polypeptide regions. In agreement with this notion, co-immunoprecipitation of preMBP or prePhoE with anti-SecB sera is only observed when SecB is present cotranslationally or upon dilution of denatured preprotein. Extensive deletion analyses of prePhoE indicate multiple (at least four) SecB binding regions in the mature protein, suggesting that SecB binds preproteins via multiple weak binding sites to unfolded regions. These interactions together result in preprotein specificity and a stable S e c B / p r e p r o t e i n complex. PhoE lacking a leader sequence is also efficiently immunoprecipitated with anti-SecB sera [124]. It appears likely that SecB interactions are substantially influenced by the folding properties of preMBP and prePhoE. As a result, association with SecB can be affected by the leader sequence, which retards preprotein folding [36]. Direct demonstration of preprotein binding to SecB

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was achieved in vitro using purified proteins. Gel filtration and density gradient centrifugation were used to isolate SecB complexes with preMBP [138], prePhoE [84,96], or proOmpA [96]. These results indicate that the S e c B / p r e p r o t e i n complex is reasonably stable under in vitro conditions. SecB (tetramer) and proOmpA form a 1:1 stoichiometric complex [82,96], which prevents aggregation of the preprotein upon dilution out of denaturant. In addition, purified SecB forms isolable complexes with mature OmpA [96] and MBP [138], showing that the leader sequence is not necessary for SecB-preprotein interactions. As observed in co-immunoprecipitation experiments discussed previously, S e c B / p r e M B P or MBP complexes are only detected when refolding of the denatured protein is initiated in the presence of SecB; no complex is observed when the protein is already folded [138]. These studies directly demonstrate that there is no specific recognition of the leader sequence by SecB and suggest that some characteristic of the unfolded or partially folded preprotein is recognized by SecB.

3.5.4. SecB preprotein recognition and binding specificity Structural studies have been carried out to investigate S e c B / p r e p r o t e i n recognition. Upon folding, preMBP displays an increase in intrinsic tryptophan fluorescence. SecB blocks this increase in preprotein fluorescence suggesting that it retards the folding of preMBP [139]. In contrast, interactions between SecB and p r o O m p A or prePhoE are not so readily apparent at a structural level. Upon dilution out of denaturant both p r o O m p A and p r e P h o E have 45%/3-sheet structure as revealed by CD spectroscopy [82,84]. The CD spectra of prePhoE diluted out of denaturant is unaffected by the presence of SecB [84]. Dilution of proOmpA or prePhoE out of denaturant in the presence of SecB also has no observable effect on the preprotein tryptophan fluorescence [82,84]. In order to examine more specific structural changes in proOmpA upon SecB binding, fluorescence energy transfer between proOmpA coumarin maleimide derivatized cysteines and tryptophan residues was measured. Fluorescence energy transfer in the presence of SecB is intermediate between that of the folded preprotein (alone in aqueous buffer) and that of the denatured preprotein [82]. These results suggest either that SecB interactions with proOmpA and prePhoE are subtle and not observed at a secondary structural level or that these interactions involve movement of secondary structural elements with respect to one another. These experiments further substantiate the difference between SecB interactions with soluble periplasmic derived preproteins and outer membrane derived preproteins. The ability of SecB to block changes in preMBP tryptophan intrinsic fluorescence, as this preprotein

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refolds, has been used as an assay to examine the specificity of SecB/mature preprotein domain interactions. SecB dissociation constants (K d) are approx. 2 nM for MBP and 50 nM for RBP, which does not require SecB in vivo [140]. Although the affinity of SecB for preMBP is quite high (similar to that of MBP), the preprotein must be released from this complex prior to or during translocation. Point mutations in SecB have been isolated, which reduce the rate of preMBP secretion in vivo [141]. These SecB mutations result in a higher affinity (by 2-3-fold) for MBP in vitro, indicating that increasing the stability of the SecB/preprotein complex can be deleterious. In addition to denatured preproteins, denatured BPTI, RNase A, and tryptophan synthase a-subunit interact with SecB as shown by their ability to compete for blockage of MBP folding [140]. These experiments illustrate the ability of SecB to bind non-native proteins with similar affinities, irrespective of whether they are secreted or cytosolic proteins. Because of this apparent lack of binding specificity, it has been proposed that selectivity in SecB binding is dictated in part by kinetic partitioning between the preprotein folding pathway and SecB association [140]. This attractive hypothesis suggests that selectivity in SecB binding is a function of the lifetime of the unfolded or partially folded protein and would be inconsistent with the existence of a specific preprotein binding 'site'. Although numerous experiments indicate that SecB recognizes nonnative structure, the question of the constraints on this recognition remains. A number of different peptides, all with a net positive charge, were shown to bind SecB, as assayed by an increase in SecB proteinase resistance [142]. These results confirm the nonspecific nature of SecB binding in vitro. Consistent with the requirement for positively charged peptides, poly-lysine and poly-arginine both protect SecB from proteolysis. The shortest peptides able to protect SecB are 14 to 15 amino acyl residues long and one mole of 260 residue poly-lysine can protect approximately 18 moles of SecB. These results indicate multiple binding sites on SecB for small positively charged peptides. The fluorescent dye ANS has been used to probe for hydrophobic regions on SecB [142]. ANS binds hydrophobic protein patches and displays a characteristic fluorescence. While neither free polypeptides nor free SecB display any ANS binding, the presence of polypeptides and SecB results in substantial ANS binding. Although it is not possible from these experiments to determine if ANS is bound to SecB, polypeptides, or both, it is attractive to speculate that upon ligand binding, SecB exposes hydrophobic patches that may ultimately be involved in preprotein binding. This work reinforces the notion that SecB interacts with preprotein via multiple low affinity binding regions. These multiple interactions could result in the required affin-

ity and stability of the SecB/preprotein complex and could also provide a basis for binding specificity.

3.5.5. SecB-dependent and -independent preproteins Despite the progress made in delineating SecB-preprotein interactions, there is not at present a satisfying explanation for SecB preprotein specificity in vivo. For example, both non-secretory preproteins and SecBindependent preproteins can require SecB under specific conditions [143,144] suggesting that the distinction between SecB- dependent and independent preproteins is not absolute. SecB specificity in vivo is not dictated by preprotein leader sequence [145] and studies of preprotein chimeras suggest that the first third of MBP dictates SecB-dependence [146,147]. A better understanding of preprotein folding should provide further insights into SecB specificity in vivo. 3.5.6. SecB preprotein targeting SecB functions in the early stages of preprotein translocation via its interaction with the preproteins. These interactions prevent misfolding and aggregation in the case of outer membrane proteins and tight folding in the case of periplasmic proteins. By preventing such non-productive states of preproteins, SecB is involved in targeting preproteins to the membrane, because it increases their functional resident time in the cytosol. Indeed, a reduction of preLamB binding to IMV is observed in the absence of SecB [148]. A direct role for SecB in preprotein targeting is suggested by binding experiments [149] which showed that SecBproOmpA complex binds to IMV (Ko 6" 10 -8 M) and that this saturable binding requires SecA. SecB bound to IMV stabilizes proOmpA for translocation [149], suggesting that this chaperone can also function bound to the membrane. Furthermore, the presence of proOmpA increases the binding of SecB for IMV by 2-3-fold. These results are consistent with the in vitro observation [149] that the presence of SecB not only reduces nonspecific binding of proOmpA to IMV but increases the productive binding of the preprotein. SecB stabilizes preproteins in a translocation competent state by direct binding interactions. As a result of these interactions with SecB, nonproductive states are prevented and therefore translocation efficiency is increased. This model suggests that SecB should not increase the rate of translocation under optimal conditions (translocation competent preprotein at saturating concentration) although the apparent rate of translocation should increase with subsaturating levels. In addition, SecB-preprotein complexes can bind SecA either at the cytoplasmic face of the membrane or in the cytosoi [149]. Consequently, SecB-preprotein interactions position the preprotein at the cytoplasmic membrane in a translocation competent conformation. The

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next section will address how the preprotein crosses the lipid bilayer.

4. Preprotein translocation across the cytoplasmic membrane How do proteins, with many hydrophilic residues, cross the hydrophobic cytoplasmic membrane to reach their final periplasmic or outer membraneous destination? Genetic and biochemical studies have identified and characterized the central components (Table 3) involved in protein secretion in E. coli. Membrane associated proteins, integral membrane proteins and acidic phospholipids comprise the translocation apparatus which will be discussed in the first section. Preprotein translocation requires energy derived from A T P hydrolysis and the transmembrane electrochemical gradient of protons (A/xH+). The second section will emphasize studies on the respective contribution of these two energy sources to the translocation process. Finally, a third section will address the question of the immediate environment of the preprotein while crossing the cytoplasmic membrane.

4.1. Translocation apparatus 4.1.1. Genetic identification Three genetic screens, based on different strategies, have been used to identify genes encoding components of the protein secretion pathway (for review see [150]). Recessive conditional lethal mutations resulting in a generalized secretion block (sec) and dominant mutations affecting protein localization (prl) have been isolated. Some prl mutations were found to be allelic to sec mutations prlA /secY, prlG /secE and prlD / secA.

Table 3 Proteins involved in translocation Protein Gene Map position SecB DnaK/DnaJ GroEL/GroES Ffh/Ffs SecA FtsH SecD SecE SecF SecY Bandl p12 Leader peptidase Signal peptidase II

secB dnak/j groEL/ES ffh/ffs secA fish secD secE secF secY ? ?

lepB lspA

81 0.5 94 57/10 2.5 69 9.5 89 9.5 72 ~ 69 55.5 0.5

a C, cytosolic;CM, cytoplasmic membrane. b Apparent Mr.

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Depending on the length of the mature portion of the preprotein, a fusion protein (consisting of the cytoplasmic enzyme LacZ connected to the amino-terminus of a preprotein) is either localized to the cytosol or becomes jammed in the membrane [40]. Consequently, the level of LacZ activity varies, and is low when the fusion protein is membrane localized [41]. A strain with such a fusion protein is unable to utilize lactose as a sole carbon source (Lac-). Selection for Lac + phenotype yielded recessive conditional lethal mutants unable to target the fusion protein to the membrane. Mutations were identified in secA [15l] and secB [120] which resulted in pleiotropic secretion defects. Using the same approach with other preprotein-LacZ fusions, the secD locus was identified [152]. Further characterization of secD [153] showed that at least two genes, secD and secF, are co-transcribed in this operon. Alteration of leader sequences of exported proteins prevent their secretion and thus their correct localization, but not their synthesis [154]. The rational of another screen [154-156] was to isolate extragenic suppressors for preproteins with altered leader sequences, which resulted in their export. This approach was used successfully with leader sequences mutations in preLamB [155] and preMBP [156]. Strains possessing a mutant lamB are unable to grow on maltodextrin as a sole carbon source ( D e x - ) and selection for Dex + pseudorevertants led to the isolation of secretion mutants. A m o n g these suppressors, prlA [155] was demonstrated to be allelic to secY. In a similar screen, starting with strains possessing a mutant malE, prLA (secY) and prlD (secA) [156,157] were identified by growth on maltose as a sole carbon source. It must be noted that these screens used deletion mutations in leader sequences in order to prevent true revertants. As a result, it is difficult to imagine, that the isolated suppressors are allele specific. In fact, it has been demon-

Mr

Location "

Suggestedfunction

Ref.

17 69/41 57/10 50/4.5S 102 71 65 14 35 49 15 ~ 12 36 18

C C C C C/CM CM CM CM CM CM CM CM CM CM

secretory chaperone general chaperone general chaperone chaperone translocase(ATPase) unknown late stages translocation translocase late stages translocation translocase translocase translocase maturation maturation

59,118-121,129-132 86-88,380-383 86-89,375-379,391 109,113 151,156,169-171,196,199,200, 202 185-188 60,152,179 181,193 158, 160,164, 165,175,178 60, 152, 179-181,193 155,156,160,175,177,384-386 175, 183 184 278, 294-298, 303 294, 387-389

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strated that some prl mutations allow secretion of leaderless preproteins [69] consistent with these prl mutations being bypass suppressors. Using the same approach but starting with a single base change mutation in preLamB, an additional suppressor class priG, shown to be allelic to secE, was isolated [158]. It should be noted that neither secB nor secD was identified by this leader sequence suppressor screen. On the basis of the observation that secA is derepressed in cells with a defect in protein secretion, t h e strategy for the third approach used secA-lacZ as a reporter. When protein secretion is impaired, expression of SecA-LacZ increases, giving rise to increased LacZ activity [159]. Mutations were obtained in secA, secD, secY and secE [160]. SecY, secE and secD were identified in two screens and secA in all three screens, suggesting that the central components of the secretion pathway have been identified. Nonetheless, additional screens making use of less well characterized preproteins, such as preBla or preRBP, would be necessary to confirm this notion.

4.1.2. Temperature sensitiue mutants Conditional-lethal mutants sensitive to high (ts) or low (cs) temperature, isolated from the previously described screens, allowed further characterization of preprotein translocation. The ts and cs mutants are defective both for growth and protein secretion at nonpermissive temperatures, 42°C and 23°C, respectively. Ts mutations have only been isolated in secY and secA. secY24 ts (obtained by chemical mutagenesis) is defective in translocation of exported proteins, such as preMBP, preLpp, proOmpA, and proOmpF at 42°C [161]. SecA51 ts was identified as a spontaneous temperature sensitive mutant which accumulates preMBP at 42°C [151]. Further studies using strains containing secY24 or secA51, showed that leader peptidase insertion into the cytoplasmic membrane was also affected at the nonpermissive temperature [162]. These results lead to the suggestion that SecY and SecA functions are not restricted to trarlslocation of preproteins possessing a cleavable leader sequence, since leader peptidase is synthesized without a leader sequence and yet is sec-dependent for insertion. Cs mutations in secY, secE, and secD have been identified in a screen for derepression of secA. With secY39 cs, proOmpA and preMBP translocation is decreased at the nonpermissive temperature [163]. Cs mutations were also identified in secE [164]. In particular secE501 [160] exhibits a secretion defect for preMBP, preLamB, and preRBP. However, secE501 is altered in its non-coding region and results in a decrease in SecE expression [165]. SecD cs mutations (for example secD1) accumulate preMBP, preRBP and p r o O m p F at 23°C [152].

4.1.3. Biochemical identification of SecA, and S e c Y / E Analysis of preprotein translocation at the molecular level requires the purification of Sec-proteins. Initially, an assay was developed which measured the post-translational translocation of in vitro translated radiolabeled preproteins into inverted cytoplasmic membrane vesicles, IMV [166-168]. Preprotein translocation was assessed by the proteinase inaccessibility of the preprotein which had translocated into the lumen of IMV (Fig. 2). Two different approaches were used to assay SecA function, complementation of secA ts and the other approach took advantage of the peripheral nature of SecA association with IMV (discussed later). Translocation of prePhoA, proOmpA, and proOmpF-Lpp is not observed in IMV derived from a secA51 ts strain [169,170] or IMV washed with urea [170,171]. In both cases, addition of purified SecA [169-171], restores preprotein translocation activity, demonstrating that SecA is strictly required for the translocation process. Consistent with the in vivo requirement of SecY for protein secretion, proOmpA translocation into IMV derived from a secY24 ts strain is defective, whereas

Protease ~ Treatment

Intermediate

Fully translocated

Fig. 2. Proteinase inaccessibility assay of translocation. Radiolabeled preprotein is added to IMV in the presence of SecA and ATP. After incubation at 37°C, the reaction is chilled on ice and proteinase is added. Untranslocated preprotein is proteolyzed, whereas the unaccessible portion of the partially translocated preprotein (intermediate) and the fully translocated preprotein are protected from proteolysis. The protected preprotein and preprotein fragments can then be visualized by SDS-PAGE subjected to audioradiography.

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translocation is restored in a similar strain carrying wild type secY on a plasmid [172]. Further confirmation of the role for SecY in preprotein translocation came with the solubilization and reconstitution of I M V proteins in proteoliposomes [173,174]. The ability to solubilize and reconstitute I M V proteins into translocation competent proteoliposomes enabled the functional purification of SecY in association with SecE [175]. Purified S e c Y / E proteoliposomes function catalytically, i.e., catalyze multiple rounds of translocation, per active S e c Y / E site, with p r o O m p A as a substrate [176]. SecY and SecE were also simultaneously overproduced and separately purified [177,178]. Optimal translocation of p r o O m p A and a truncated p r o O m p A derivative requires the presence of both SecY and SecE [177,178]. Together these results establish the essential role of S e c Y / E in preprotein translocation. In addition to SecA and S e c Y / E , other less characterized integral m e m b r a n e proteins have been implicated in preprotein translocation, such as SecD, SecF, Band 1, and p12. Biochemical studies [179-181] suggest that SecD and SecF are involved in the later steps of preprotein translocation (discussed elsewhere). In addition, SecD and SecF have been purified from an overproducing strain, although no translocation activity was observed [182]. Band 1 was identified as a polypeptide which associates with SecY and SecE, during both purification [175] and immunoprecipitation with antiSecY sera [183]. Another polypeptide, termed p12, has been recently purified from a T C A soluble fraction of

323

solubilized cytoplasmic m e m b r a n e s [184]. Stimulation of p r o O m p F - L p p and p r o O m p A translocation is observed when p12 is reconstituted in the presence of SecY and SecE into proteoliposomes [184]. Nevertheless, the functions of both Band 1 and p12 remain unknown. The similar size of these two polypeptides suggests that they may be identical. Recent studies have implicated an additional protein, FtsH [185], in protein assembly into and translocation across the cytoplasmic m e m b r a n e [186-188]. Mutations in ftsH result in a defect in the assembly of penicillin-binding protein 3 in the cytoplasmic m e m b r a n e [186] along with a reduction in the translocation of preBla [187,188]. Further studies on FtsH will be required to elucidate its roles in preprotein translocation and protein insertion. 4.1.4. S e c Y / E / A organization and function SecY and SecE interact functionally and physically as demonstrated both by in vivo and in vitro studies. Overproduction of secY is dependent on the simultaneous overproduction of secE, yet not the converse [189,190]. SecY and SecE co-chromatograph and coimmunoprecipitate [175]. The dissociation of the S e c Y / E complex at 20°C correlates with a loss in p r o O m p A translocation-dependent activity [183]. Genetic and biochemical investigations have demonstrated a molar excess of SecE to SecY (between 2:1 and 5 : 1) [176,182,191,192]. Genetic analyses, using suppressor-directed inacti-

A D P + Pi

ATP

SecA Cytoplasm -

OH-

SecY

.,cE +

H +

Periplasm Fig. 3. Minimal components required for preprotein translocation. The integral membrane proteins SecY and SecE along with acidic phopholipids and the peripheral membrane protein SecA are absolutely necessary for preprotein translocation. In addition, energy derived from ATP hydrolysis and a zi/zH+ drives the preprotein across the membrane. SecB, SecD, SecF, and leader peptidase are not shown as these proteins are not absolutely required for preprotein translocation. Furthermore because the roles of band 1 and p12 are unknown they have not been included.

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vation and Sec-titration, suggest that SecY and SecE interact directly during preprotein translocation [191,193]. Suppressor-directed inactivation results from interactions between defective leader sequences and SecY or SecE altered by suppressor prl mutations [191]. By combining this technique with conditional lethal sec mutations at semi-permissive temperature (refered to as Sec titration), further support for a translocation complex containing SecY and SecE was obtained [193]. From Sec titration experiments, it has been suggested that SecY and SecE assemble and disassemble during the translocation process [191,193]. Although these genetic data are fully consistent with such a hypothesis, caution must be taken in interpreting results using multiple mutants of a multisubunit protein. However, SecY/E complexes appear to be stable once formed, inconsistent with a cyclical assembly mechanism [190]. Biochemical experiments are clearly required to directly address this issue. In vitro, addition of SecA suppresses the proOmpA translocation defect observed in IMV derived from secY24 ts, leading to the suggestion that SecA interacts with SecY [194]. Furthermore, SecA binds urea-washed IMV with high affinity (K d 4.10 -s M) and preincubation of IMV with anti-SecY sera decreases binding by 3-fold [149]. Finally, the presence of SecA protects SecY against limited proteolysis [149]. These results indicate that SecA associates with SecY/E. On the basis of functional and physical interactions, the multisubunit enzyme SecA/SecY/SecE has been refered to as translocase (Fig. 3). In the following sections, specific studies on SecA, SecY/E, and additionally acidic phospholipid involvement in preprotein translocation will be discussed. For specific reviews on SecY, SecA and acidic phospholipids, see Refs. [195, 196,197], respectively. 4.1.5. SecA E. coli secA encodes a peripheral [159] membrane ATPase [198,199] of 901 amino acids [200]. SecA is

conserved in other prokaryotes with homologs identified in Bacillus subtilis and Bacillus licheniformis [201]. SecA ATP hydrolysis is essential for preprotein translocation [198] and in vitro measurements of SecAdependent ATP hydrolysis have enabled a detailed examination of the requirements for this reaction. SecA exhibits different levels of ATPase activity: a low endogenous ATPase activity, stimulated by liposomes (termed lipid-ATPase [202]) and further stimulated by translocation components (termed translocation ATPase [199]). In addition, SecA binds both preproteins and SecB, and thus has been suggested to function as a receptor during preprotein secretion [148,149]. This section will explore the interactions of SecA with preproteins, SecB, and membrane components. Treatment of IMV with azido-ATP and UV irradia-

tion inactivates membrane vesicles for preprotein translocation [203], suggesting that the requirement for ATP in preprotein translocation is mediated by a membrane-associated protein. Purified SecA restores proOmpA translocation in these azido-ATP photoinactivated IMV [199], demonstrating that SecA has a central role in coupling ATP hydrolysis to preprotein translocation. Examination of the SecA sequence reveals several putative ATP binding sites [204,205]. One of these sites, corresponding to a conserved Walker nucleotide binding motif, was mutated in the Bacillus subtilis secA (which is 53% identical to E. coli secA). This mutation destroys the ability of B. subtilis secA to complement E. coli secA ts [205] and abolishes preprotein-dependent ATP hydrolysis [206]. In E. coli SecA, two ATP binding motifs have been shown by site directed mutagenesis to be essential for SecA-dependent translocation ATPase and in vitro preprotein translocation activities [393]. Derivatization of purified SecA with 8-azido-[32p]ATP (12 raM) results in two moles of azido-ATP crosslinked to a mole of SecA [199]. Photoaffinity crosslinking studies with [32p]ATP (0.1 mM) was used to localize an ATP binding site within a 4 kDa region residing in the amino-terminal 217 residues of SecA [207]. In uiuo, sodium azide inhibits the export of preMBP and proOmpA [208] and isolated azide resistant mutants [209] were shown to be alleles of secA [208]. In vitro, sodium azide also inhibits proOmpA translocation into IMV, suggesting that sodium azide affects preprotein translocation in vivo via SecA [208]. Both these in vitro and in vivo studies indicate that preprotein translocation requires SecAdependent ATP hydrolysis. Optimal SecA-ATPase activity (translocation ATPase) requires both SecY and a translocation-competent preprotein [199]. Membrane-bound SecA ATPase activity is increased up to 20-fold with translocation-competent proOmpA and to a lower extent with prePhoE [198,199]. This stimulation of SecA ATPase activity is not observed with IMV derived from a secY ts strain nor with IMV pretreated with anti-SecY sera [199]. Consistent with the requirement for a translocation-competent preprotein, neither the mature domain of proOmpA nor the leader sequence, separately, are able to stimulate SecA-ATPase activity [198]. However, functional leader peptides competitively inhibit proOmpA-dependent SecA-ATPase activity [198]. These studies suggest that the leader sequence is directly recognized by SecA. Additionally, the preprotein mature portion was also shown to be required for the stimulation of SecA-ATPase activity, using aminoterminal fragments of proOmpA [64] and this will be discussed elsewhere. Direct demonstration of SecA/preprotein interactions came from binding analysis [149] and chemical crosslinking studies [210,211]. Scatchard analysis shows

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both a high affinity SecA-dependent binding of SecB (2" 10 -7 M) and of proOmpA (6" 10 8 M) to IMV [149]. SecA-dependent binding of SecB and proOmpA is saturable, with approximately 100 pmol binding sites per mg of IMV protein [149], comparable to the amount of SecY in IMV [176,182]. Together these data are consistent with SecA functioning as a peripheral membrane receptor for both SecB and preprotein. Further support for SecA/preprotein interactions came from chemical crosslinking studies which show that the leader sequence is necessary for preprotein SecA crosslinking [210] and that the mature domain of proOmpA is proximal to SecA during preprotein translocation ([211], discussed elsewhere). In addition, crosslinking studies using SecA reconstituted from amino- and carboxy-terminal fragments have identified a region of SecA between amino acid residue 267 and 340 [212] involved in SecA-preprotein interactions, which is adjacent in primary sequence to an ATP binding site. SecA lipid-ATPase is a model reaction that has been used to examine ATP-dependent SecA-preprotein interactions in the absence of translocation. SecA lipid-ATPase stimulation is observed with the simultaneous addition of the leader and mature parts of proOmpA or preMBP [202]. These results suggest that SecA can recognize both the leader peptide and the mature domain of the preprotein separately. Furthermore, the addition of ATP, but not AMP-PNP, to liposomes-bound SecA results in the release of bound proOmpA from SecA [77]. This observation led to the proposal that the preprotein binds SecA upon binding ATP, and ATP hydrolysis specifically results in preprotein dissociation [77]. Conversely, proOmpA addition to SecA in solution results in the release of bound ADP [213]. By extrapolation to the translocation process, cycles of ATP-dependent SecA preprotein binding and release could facilitate preprotein movement across the membrane. Biophysical studies on SecA interactions with either lipid monolayers or liposomes indicate that SecA inserts into phospholipid membranes. An increase in lipid monolayer surface pressure is observed with either SecA alone or SecA and a non-hydrolyzable ATP analog, indicative of SecA membrane insertion [214]. In the presence of ADP, SecA does not insert into lipid monolayer, suggesting that nucleotide hydrolysis reverses insertion [214]. Furthermore, studies using quenching of SecA intrinsic fluorescence by brominated or spin-labeled phospholipids in model membrane vesicles, show that SecA inserts deeply into the phospholipid bilayer [215]. In addition, SecA causes an increase in steady state fluorescence anisotropy of a membrane probe embedded in DOPG vesicles, interpreted as an insertion of SecA into the lipid acyl chain region [216]. Using this technique, no insertion of SecA is observed in the presence of ADP. Together, these

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model studies raise the attractive possibility that SecA membrane insertion is regulated by binding and hydrolysis of ATP. In addition, SecA insertion into acidic phospholipid vesicles occurs concomitant with an increase in proteinase sensitivity and a decrease in the thermal transition of unfolding, indicative of a major conformational change [215]. Changes in soluble SecA proteinase sensitivity in the presence of ATP, preprotein or IMV are also observed [213], indicative of conformational changes. Addition of ATP results in an increase in proteinase resistance of soluble SecA, whereas the addition of preprotein, IMV or acidic phospholipids renders SecA more proteinase sensitive [213]. Although these model reactions provide insights into SecA interactions with preprotein, membrane bilayer and nucleotides, the functional role of SecA membrane insertion and its concomitant conformational changes in preprotein translocation has yet to be determined. SecA is found both associated with the cytoplasmic membrane and soluble in the cytosol [217], indicating perhaps that SecA has more than one function in protein secretion. Cell fractionation [217], gel filtration [218], chemical crosslinking experiments [210], and electron microscopy of three-dimensional crystals [219] of SecA all indicate that the predominant form of soluble SecA is dimeric. Recent studies suggest that the dimeric form of SecA is functional for proOmpA translocation [220]. Inactivation of one subunit of the SecA dimer results in loss of proOmpA translocation activity. The addition of this inactive heterodimer to a functional homodimer does not result in a further decrease in translocation activity, suggesting that SecA subunits do not exchange during translocation. It is possible that oligomeric states of SecA are involved in the regulation of preprotein translocation. In vivo, SecA synthesis is regulated by preprotein secretion. Derepression of SecA occurs when secretion is compromised. Specifically, mutations in the membrane associated Sec-proteins [221], reduction in acidic phospholipid levels [222], or synthesis of preMBP-LacZ chimeras, which blocks secretion [159], all derepress SecA synthesis. Regulation of SecA synthesis has been shown to occur at the translational level [223], although SecA was found to bind a variety of preprotein mRNA with the same affinity as its own mRNA [224]. The translational regulation of SecA synthesis by the secretion process provides a simple means for the control of protein secretion although the precise mechanism of this regulation is not yet elucidated. 4.1.6. SecY and SecE A large number of genetic and biochemical experiments demonstrate that SecY is an indispensable component of the secretion machinery. As discussed previously, two different selection screens yielded two classes

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porting the proposed topology of SecY [225]. E. coil secY homologs have been identified in a large variety of prokaryotes, such as B. subtilis [229,230], B. licheniformis [231], Staphylococcus carnosus [231], Lactococcus lactis [232], Microccocus luteus [233], Methanococcus vannielii [234]. Analysis of hydropathy profiles and alignment of secY sequences suggest that SecY membrane topology (with 10 transmembrane domains) is conserved. More than 30 prl mutations have been characterized in E. coli secY and at present, 25 of these mutations have been sequenced [235-240]. These mutants were isolated by their ability to export either preLamB or prePhoA with an altered leader sequence hydrophobic core, or preMBP with an altered charge distribution near the leader sequence. Prl mutations are not randomly distributed throughout SecY, suggesting that their clustering has some functional significance [239]. Specifically, most mutations map to the first periplasmic loop (P1), the seventh transmembrane domain (TM7) and the tenth transmembrane domain (TM10). These mutations might either be directly involved in leader sequence recognition or may bypass

of secY mutants, those defective in protein secretion [161] and those with altered substrate specificity [155]. These loss of function (sec) and change of function (prl) mutants combined, suggest that SecY is a central component of the preprotein translocation apparatus. Involvement of SecY in translocation was also established in vitro, using specific antibodies [225,226] and purification/reconstitution techniques [175,177], as discussed previously. The translocation activity of SecY requires the presence of another integral membrane protein SecE [175,177], and different experiments indicate functional and physical interactions between SecY and SecE [175,190,191]. We shall examine, in the subsequent sections, the functional domains of these components as suggested by mutational analyses. E. coli secY encodes an integral membrane protein of 443 amino acids [227]. On the basis of both proteolysis and TnphoA transposition, a topology for SecY (Fig. 4) was proposed consisting of 10 transmembrane domains, with its carboxy-terminus and amino-terminus residing in the cytosol [228]. Antibodies directed against the carboxy- and amino-terminus of SecY, were shown to interfere with binding of preprotein to IMV, supP1

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R.A. Arkowitz, M. Bassilana/ Biochimica et BiophysicaActa 1197 (1994) 311-343 the requirement for leader sequences in translocation. Recent work [69] favors the latter interpretation, by demonstrating that prl mutants are able to export both MBP and PhoA lacking leader sequences. In addition, one of the strongest prl mutations is located in the first periplasmic loop of SecY [238] and two other prI mutations result in a large deletion in TM7 domain [239], consistent with the notion of prl mutations bypassing the leader sequence requirement. Three conditional lethal secY mutations have been mapped, two of these to P5 (secY39 cs and secY40 cs, [163]) and one to P4 (secY24 ts, [161]). The effects of two of these single amino acid changes on SecY function have been examined both in vivo and in vitro. SecY39 cs results in almost a complete block of proOmpA and preMBP secretion at 23°C. In vivo, secY39 cs responds rapidly to a decrease in temperature, with preprotein accumulation as early as 1 min, after a shift to 23°C [163]. In vitro, this mutation also confers a strong preprotein translocation defect. In contrast, secY24 ts requires greater than an hour at the nonpermissive temperature (42°C) to display a protein secretion defect [161,162]. The in vitro secretion defect of secY24 can be overcome by increased levels of SecA [194], suggesting that this region may not be directly involved in preprotein translocation. Using secY mutants and secY-phoA fusions that interfere with protein secretion in wild type cells, a region in SecY that may interact with other translocation components has been delineated [241]. PhoA fused to SecY at P4, but not at the amino-terminal portion of P3, inhibits preMBP secretion in wild type cells in vivo. It has therefore been suggested that a region of SecY maximally extending from P3 to TM7 confers this trans-dominant effect. Overproduction of SecE in the presence of these dominant secY-phoA fusions abolishes the protein secretion defect, indicating that the P3 to TM7 region of SecY interacts with SecE. A secY trans-dominant mutant has also been isolated by its ability to inhibit protein secretion in wild type cells. This mutation (secY al) maps to TM9 and results in a 3 amino acid deletion [241]. It was postulated that this mutation abolishes SecY translocation function, yet not its ability to interact with SecE, resulting in the sequestering of SecE in a dead-end complex. As expected, SecE overproduction rescues the trans-dominant negative phenotype of secY dl. These studies are consistent with the region P3 to TM7 interacting with SecE, yet need to be substantiated at a biochemical level. A specific interaction between SecE and SecY has been inferred by the lethal phenotype of the combination of two prl mutations, prlA4 and priG1 [191]. Both of these mutations alone result in the ability to secrete preproteins with mutant signal sequences. Together, these change of function mutations confer a recessive allele specific lethality, indicative of a direct interaction

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between SecY and SecE, that in this case results in an inactive complex (dead-end). A coherent picture of the different functional domains of SecY will require extensive biochemical analyses of change of function mutants and loss of function mutants. Notably, secY mutations obtained in E. coli, do not map to conserved residues among secY homologs [231]. Swapping of SecY domains between E. coli and other prokaryotes, may be useful in establishing the functional regions in SecY. SecE is an integral membrane protein of 127 amino acids which has been proposed, based on TnphoA analysis, to span the cytoplasmic membrane three times with its amino-terminus facing the cytoplasm [164]. Based on the expression levels of SecE-PhoA fusions, the cellular content of SecE was estimated to be 250500 molecules/cell [165]. At present, six cs mutations in secE have been identified and all reduce SecE expression approximately two-fold [165]. It appears that protein secretion is sensitive to SecE levels in uiuo, especially at low temperature. SecE is essential for cell survival [165]. The third transmembrane domain of SecE complements a secE cs mutant resulting in normal levels of protein secretion [165,192]. Purified SecE carboxy-terminal half (including the third transmembrahe domain), when reconstituted into proteoliposomes, is active in translocation to a similar extent as full length SecE [192]. In addition, this carboxyterminal fragment of SecE allows the overproduction of SecY [192]. All priG mutations are located in the third transmembrane domain or the periplasmic carboxy-terminus. Together these results suggest that the third transmembrane domain of SecE, in particular, interacts with SecY. Homologs of E. coli secE have also been identified in B. subtilis and B. licheniformis [242]. These SecE homologs appear to have only one transmembrane domain, which is homologous to the carboxy-terminus of E. coli SecE. This conserved region further indicates that E. coli SecE function may require only the third transmembrane domain of this protein.

4.1.7. Acidic phospholipids E. coli membranes are composed of approximately 75% of the neutral phospholipid, phosphatidyl ethanolamine (PE), and 25% acidic phospholipids, mainly phosphatidylglycerol (PG; 20%) and cardiolipin (CL; 4%) [243]. Involvement of acidic phospholipids in preprotein translocation was initially demonstrated [244], using a strain in which the level of acidic phospholipids can be manipulated by controlling the activity of phosphatidyl glycerol phosphate synthase [245]. In vivo, the rate of maturation of p r o O m p A and prePhoE is reduced when membrane PG levels are lowered. Consistently, the rate of prePhoE translocation is reduced 2-fold in IMV depleted of PG and CL. These results

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demonstrate that acidic phospholipids are involved in preprotein translocation both in vivo and in vitro. Confirming this initial characterization, translocation of proOmpA is also impaired in IMV with reduced PG levels [202] and in proteoliposomes reconstituted with SecY/E and only the neutral lipid DOPC [246]. In contrast, proOmpA translocation takes place in proteoliposomes composed of SecY/E and DOPG [246]. Moreover, prePhoE and proOmpA translocation is restored upon readdition of acidic phospholipids to IMV depleted of PG [246,247]. PrePhoE translocation activity increases 4-fold when the PG content of IMV increases from 5 to 20% [247]. This stimulation is independent of the nature of the acidic phospholipid added, suggesting that the negative charge of the head group is the determining factor in restoring prePhoE translocation. The requirement for acidic phospholipids in preprotein translocation may be indirect, through interactions with the translocation components SecA or SecY/E, or direct, through interactions with the preprotein (via the leader sequence). Consistent with an effect of acidic phospholipids on SecA activity, SecA translocation ATPase activity and SecA-lipid ATPase activity are dependent on the presence of acidic phospholipids [202]. Translocation ATPase is reduced 12-fold in IMV containing low levels of PG and CL, and SecA-lipid ATPase is reduced 7-fold in liposomes constaining only DOPC. The reduction of SecA ATPase activity in the absence of acidic phospholipids reflects the inability of SecA to specifically bind PG-depleted IMV [246]. In agreement with these in vitro results, less SecA fractionated with IMV derived from a PG-depleted strain compared to a wild type strain [217]. However, it is not possible to assess whether acidic phospholipids participate directly in high affinity SecA binding or exert their effects indirectly via an effect on SecY/E stabilization. SecA has been shown by a number of techniques to bind directly to acidic phospholipid containing liposomes. Using flotation gradient centrifugation SecA was shown to bind liposomes containing PG and CL, but not DOPC and DOPE [202]. In addition, binding of SecA to liposomes, as assayed by fluorescence quenching experiments, increases from 20 to 80% with D O P C / D O P G vesicles compared to DOPC vesicles [215]. Together, these results indicate that SecA can bind directly to acidic phospholipids, possibly via elecTable 4 Driving forces for preprotein translocation (1) (2) (3) (4)

ATP binding and hydrolysis Transmembrane chemical proton gradient Transmembrane electrical gradient Other less characterized driving forces such as protein unfolding/folding

The contribution of these driving forces are discussed in section 4.2.

trostatic interactions. Not surprisingly, SecA insertion and concomitant unfolding in a model monolayer also requires acidic phospholipids [214,215]. Similarly, conformational changes in SecA, as assayed by proteinase susceptibility, occur in the presence of liposomes containing PG and CL, but not PE or PC [213]. These results suggest that acidic phospholipids, in combination with SecY/E, may be involved in targeting SecA in vivo. Unfortunately, because of their requirement for SecA binding, a direct role of acidic phospholipids in preprotein translocation cannot be assessed. However, an interaction between acidic phospholipids and preproteins has been suggested based upon the requirement of acidic phospholipids for the bilayer insertion of leader peptides (see previous section). For example, prePhoE leader peptide interactions with lipid monolayers depend upon acidic phospholipids [26], similar to the dependence of prePhoE translocation in vivo and in vitro [244]. Furthermore, proOmpF-Lpp translocation dependence on acidic phospholipids varies with the composition of its leader sequence [35]. ProOmpFLpp leader sequence was altered by varying the length of the hydrophobic core, by introduction of 8 or 9 Leu (8L or 9L), and by varying the number of amino-terminal positive charges, by introduction of 0 or 2 Lys (OK or 2K) [248]. Translocation of 2K8L proOmpF-Lpp leader sequence mutant increases 10-fold with an increase of PG content in IMV. In contrast, translocation of 0K9L and 2K9L proOmpF-Lpp leader sequence mutants is not dependent on PG content in IMV [35]. These results indicate that the PG requirement for translocation can be overcome by an increase in hydrophobicity of the leader sequence. These data are inconsistent with acidic phospholipids only functioning via their interactions with SecA. We have discussed in vivo and in vitro experiments, demonstrating the requirement for SecA, SecY, SecE and acidic phospholipids in preprotein translocation. Among these components, SecA plays a central role by virtue of its ability to hydrolyze ATP, providing a driving force for preprotein translocation.

4.2. Energetics of preprotein translocation Preprotein translocation is an energy-driven process (Table 4), which requires two different energy sources: ATP hydrolysis by SecA and the transmembrane electrochemical gradient of protons (A/xH+). A/xH + is strictly required for preprotein translocation in vivo, whereas ATP hydrolysis is the only energy source strictly required for in vitro preprotein translocation, with A~xH+ having a stimulatory effect. This section will discuss the relative contributions of these two energy sources in the translocation process and their possible interrelationships (for recent reviews see [249-251]).

R.A. Arkowitz, M. Bassilana /Biochimica et Biophysica Acta 1197 (1994) 311-343 4.2.1. A TP requirement

The requirement for ATP during preprotein translocation cannot be examined in vivo since it is also required for protein synthesis. As a result, the essential role of ATP in preprotein translocation was demonstrated using an in vitro translocation system, consisting of translated preprotein and IMV lacking the F1F0-ATPase [166]. In vitro translocation of prePhoA and proOmpA strictly requires ATP [166]. A ~ H + further stimulates A T P - d e p e n d e n t translocation of proOmpA [168,252], and proOmpF-Lpp [253], suggesting that these two energy sources act at different stages. Preprotein translocation can be divided into at least three stages: the initiation, as measured by the processing of the preprotein to its mature form (this would require the preprotein leader peptidase cleavage site to cross the membrane), the intermediate stage, as defined by kinetic translocation intermediates that have partially crossed the membrane, and the final stage, wherein the preprotein completes translocation and becomes totally inaccessible to added proteinase (Fig. 2). Hydrolysis of ATP is absolutely required for the overall translocation process, since non-hydrolyzable ATP analogs, such as AMP-PNP, cannot substitute for ATP and even inhibit the ATP-dependent preprotein translocation [166]. AMP-PNP binding is nevertheless sufficient to drive the translocation of a small domain of the preprotein, i.e., initiation of translocation, since maturation of proOmpA to OmpA is observed in the presence of AMP-PNP [77]. As mentioned in an earlier section, SecA insertion into a lipid monolayer can be affected by ATP binding and hydrolysis [214]. Taken together these results raise the possibility that ATP binding causes the vectorial movement of SecA into the lipid bilayer, thereby facilitating limited forward translocation of the preprotein. Isolation of proOmpA translocation intermediates allows characterization of later stages of translocation. Kinetic intermediates of translocation can be generated with micromolar concentrations of ATP [77,254]. These intermediates are the result of partial translocation of the preprotein across the membrane and can be visualized after proteinase treatment (Fig. 2). Upon addition of millimolar concentrations of A T P , proOmpA can complete translocation [77,254]. Similar to the effect of AMP-PNP on the initial stage of p r e p r o t e i n translocation, non-hydrolyzable A T P analogs can drive a limited forward translocation (2 kDa) of translocation intermediates [77,78]. These observations show that limited forward translocation driven by ATP binding energy can occur at different stages of translocation of the polypeptide chain, perhaps also due to the vectorial movement of SecA with respect to the membrane bilayer. In contrast to overall

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translocation, which appears to be an irreversible process, partial translocation is reversible during the intermediate stage. When a translocation intermediate is depleted of ATP or when SecA is inactivated, reversal of proOmpA translocation by 10 kDa is observed [77]. These results indicate that SecA and ATP are not only necessary for forward translocation, but also prevent reversal of translocation. The generality of this conclusion will require the characterization of additional preprotein intermediates. ATP hydrolysis is absolutely required for preprotein translocation across the membrane, yet the ATP hydrolysis during in vitro translocation is not coupled to net preprotein chain movement. Crosslinking of the covalently folded domain BPTI to a cysteine residue in carboxy-terminal portion of proOmpA, blocks translocation of this chimera at the BPTI moiety [77]. The rate of ATP hydrolysis remains constant when translocation of proOmpA-BPTI is blocked. In contrast, ATP hydrolysis is no longer observed when the preprotein completes translocation subsequent to cleavage of the crosslinker between proOmpA and BPTI [77]. These results are inconsistent with the existence of a strict coupling between chain movement and ATP hydrolysis and led to the proposal that futile ATP hydrolysis occurs upon ATP-dependent binding and release of p r o O m p A [77]. In agreement with this hypothesis, the amount of ATP hydrolyzed during translocation varies in a nonproportional fashion with the amino acyl length of the mature domain of the preprotein. Reduction of p r o O m p A size from 346 amino acids to 74 amino acids (including the leader sequence) leads to a reduction in ATP hydrolysis of greater than 98% [64]. Since the rates of translocation of proOmpA and of its shorter derivative are comparable, ATP hydrolysis per mol of preprotein translocated is reduced by more than 50fold, when the size of the preprotein is reduced 5-fold [64]. These results again point to the absence of strict coupling between ATP hydrolysis and net chain movement in vitro. When one divides the rate of ATP hydrolysis by the rate of proOmpA translocation, the result is that greater than 1000 mol of ATP are hydrolyzed per mol of proOmpA translocated [64,77,255]. The relevance of such a high level of ATP hydrolysis is questionable in vivo. It is unlikely that a futile cycle comprised of SecA ATP-dependent binding and release of preprotein occurs in vivo. In vivo, perhaps other factors, such as AjzH +, reduce the level of ATP hydrolysis.

4.2.2. A # H + requirement 4.2.2.1. A # H + in cir,o and in citro. In E. coli, the

t r a n s m e m b r a n e electrochemical p r o t o n gradient (A/.~H +) is comprised of two components, one electrical (A~, negative inside) and one chemical ( a p H ,

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alkaline inside). In neutrophiles, such as E. coli, the magnitude of /I/xH + is about 200 mV with the major contribution from g'. A/xH + generated in aerobic bacteria through proton extrusion by the respiratory chain [256], is used for a number of cellular functions, such as substrate transport and ATP synthesis. A similar requirement for A/xH + is observed in vivo for protein secretion in E. coli [257-261] and other prokaryotes [262-266]. Specifically, dissipation of /ItxH + in E. coli results in accumulation of unprocessed preproteins, such as proOmpA, proOmpF, preLamB, preMBP, prearabinose binding protein [257-261]. Both the electrical and the chemical components of AIxH + are required for preprotein secretion [261]. As the overall preprotein translocation appears to be an irreversible process, A # H + must act kinetically rather than thermodynamically, by accelerating the slowest step of the translocation reaction. In vitro, A/xH + alone is not sufficient to drive preprotein translocation [166], but increases the rate of ATP-dependent preprotein translocation in IMV by 2-10-fold [168,252,253,267,268]. Such a stimulatory effect of A/xH + on ATP-dependent preprotein translocation is also observed in proteoliposomes reconstituted with purified S e c Y / E , in the presence of purified SecA [175,268]. When the light driven proton pump bacteriorhodopsin [175] or an artificial potassium acetate diffusion gradient [268] is used to generate al/xH + in proteoliposomes, the rate of ATP-dependent proOmpA translocation increases. These results demonstrate a direct effect of A/xH + on the overall preprotein translocation process. Numerous in vivo and in vitro experiments have established that both components of AtxH + are required for maximal stimulation of ATP-dependent preprotein translocation [253,261,268]. The effect of varying the magnitude of A/xH +, Zigt, or ApH on proOmpA translocation into S e c Y / E proteoliposomes has been examined [255] and the relationship between the magnitude of each of these driving forces and the rate of proOmpA translocation is non-linear. It can be envisioned that /ItzH + acts by increasing the number of active translocation sites or accelerates the rate of translocation per translocation site. A role of a / x H + in recruiting active translocation sites is however unlikely since A # H + has no effect on SecA-dependent proOmpA binding to S e c Y / E [268] nor on the number of functional proOmpA translocation sites [176]. The same level of stimulation of proOmpA translocation is observed upon imposition of the same magnitude of Aq* or ApH [255] suggesting that these driving forces act on a common step. Ab~H + does not affect the initiation of proOmpA translocation [269]. In contrast, imposition of a A/~H + stimulates the overall preprotein translocation and therefore must affect a latter stage of translocation. In

agreement with such a temporal effect, AtxH + alone is sufficient for the completion of translocation of kinetic intermediates [77,254]. Furthermore, a preprotein possessing a secondary structural element at its carboxyterminus, such as a disulfide bridge, is strictly dependent on the presence of AtxH + for completion of translocation [270]. These observations lead to the conclusion that ATP hydrolysis and A # H + are required at different stages of preprotein translocation. This conclusion is additionally supported by in vivo secretion studies which demonstrate that the final step in the secretion of preMBP requires A/xH + and not ATP hydrolysis [260]. 4.2.2.2. Possible roles o f At.tH +. Although the involvement of A~xH + in preprotein translocation is well substantiated, its role remains to be elucidated. At least three possible roles of zl/xH + can be envisioned. A/xH + could cause a conformational change of the translocation apparatus itself, act directly on the preprotein, or couple preprotein translocation with an energetically favorable ion flux, via an antiport. This following section will discuss the experiments addressing these different possibilities. Both in vivo and in vitro studies indicate that the level of AFxH + stimulation varies with preprotein. In vivo, dissipation of 3txH + has a more drastic effect on the secretion of pre-/3-1actamase [261] than on that of proOmpA [257]. Similarly, in vitro studies show that zl/xH + stimulates the translocation of different preproteins to various extents. For example, AtxH + is strictly required for translocation of proOmpF-Lpp [267], as opposed to its stimulatory effect on the rate of p r o O m p A translocation. T h e i n t r o d u c t i o n in proOmpF-Lpp of a mutation leading to an uncleavable leader sequence [271], changes the strict dependence on zl/xH + for the translocation of this preprotein to a requirement similar to that of proOmpA, i.e., only stimulatory [267]. Reciprocally, the presence of an intramolecular disulfide bridge in proOmpA, changes the requirement for AtxH + from only stimulatory (for proOmpA) to strictly required (for disulfide-bridged proOmpA) [270]. Furthermore, proOmpA possessing a crosslinked loop of 13 residues strictly requires A/xH + in order to complete translocation [272], as does proOmpA crosslinked to a 20 residue polypeptide [273]. These results show that introduction of structural constraints in the polypeptide chain increases the preprotein AtxH+-dependence. Unfortunately, these data do not distinguish between AIxH + acting directly on the preprotein or on the translocation apparatus. Either effect could alter the AIxH + dependency of preproteins. Nevertheless, these studies raise the interesting possibility that A~H + might act by widening a putative translocation channel. Such a widening must however be limited in size, since A # H + is unable to drive completion of translocation of proOmpA attached to

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larger structural elements such as BPTI [176] or D H F R [78]. The possibility of an electrophoretic effect of zip.H + in preprotein translocation has been envisioned, i.e., the preprotein moving across the membrane down the electrical component of zi/xH +. In such a scenario, zig' can only accelerate the transfer of negative charges, because of its polarity across the membrane. Consistently, A/zH + stimulates translocation of proOmpA with two additional negatively charged residues, inhibits translocation of proOmpA with two additional positively charged residues and has no effect on translocation of proOmpA with two additional neutral residues in the amino-terminus mature portion [51]. Inconsistent with a purely electrophoretic effect, a short derivative of proOmpF-Lpp possessing no charges in its mature domain still exhibits A/xH+-dependent translocation [54]. Moreover, in reconstituted S e c Y / E proteoliposomes, ZIg' and ApH are equivalent in accelerating the rate of proOmpA translocation [255]. Taken together, these experiments exclude a unique role for ZI/xH + in facilitating the movement of charged preprotein residues across the membrane. Nonetheless, these data do not rule out the possibility of ZIpH acting via deprotonation of basic residues on the cytoplasmic surface of the membrane and protonation after crossing the membrane. It is difficult to generalize with respect to the role of ziIxH + in the translocation of different preproteins, because it is likely that these different substrates have different rate limiting steps. In E. coli, many transporters use the energy derived from AIxH + to couple the energetically favorable entry of protons to the accumulation (symport) or the extrusion (antiport) of small molecules, such as sugars and amino acids, against their concentration gradient. Similarly, one can envision that ZIIxH + acts via a preprot e i n / p r o t o n antiport, in which translocation of preproteins is driven by the energetically favorable proton flux. Consistent with the possible involvement of proton transfer during translocation, the rate of AIxH +dependent completion of a p r o O m p A intermediate is reduced by 3-fold in D 2 0 relative to H 2 0 [268]. More directly, a proton flux associated with translocation conditions is measured in IMV derived from a strain overproducing S e c Y / E [274]. The measured proton outflow depends on the presence of proOmpA, SecA and ATP. Despite the excess of S e c Y / E , no concomitant increase in proOmpA translocation is observed, indicating that there is no direct correlation between the preprotein translocation event per se and proton flux. As a result, net proton transfer associated with the presence of proOmpA does not reflect a strict coupling between preprotein and proton movement. Consequently, no definitive evidence of the existence of a p r e p r o t e i n / p r o t o n antiport, driven by zi/xH +, has yet been reported. Testing the existence of a prepro-

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t e i n / p r o t o n antiporter will require large amounts of functional translocation apparatus in a sealed vesicular system. Taking into account the multiple effects of ZI/xH +, it is difficult to envision that zi/xH + only acts on a single step of preprotein translocation. If there is a single zi/xH + responsive component, it appears most likely that it is the translocation apparatus itself, which in turn would affect the rate limiting step of translocation. Examining the effect of A/xH + on preprotein translocation in vitro, using various SecY mutants may be revealing. To assess more directly the role of AIxH + in preprotein translocation, it will be necessary to quantitatively examine partial translocation reactions, in which chain movement is rate limiting and driven by only zip.H +. In addition, caution must be taken in comparing in vivo and in vitro effects of zi/xH + on preprotein translocation: AtxH + stimulation of preprotein translocation in vitro is most likely an underestimation, considering both the exponential relationship between preprotein translocation and magnitude of ZIp.H + in vitro [255], and the fact that the highest magnitude of ZI/xH + generated in vitro is often far below the ziIxH + observed in vivo. 4.2.3. A T P / AIx H + interrelationship Both ATP and A/xH + are essential for maximal preprotein translocation. In vitro, the relative contribution of ATP hydrolysis and A/xH + as driving forces for preprotein translocation has been examined and shown to vary with the preprotein and the stage of translocation. This section will discuss the effect of A/xH + on S e c A / A T P driven preprotein translocation and of ATP and SecA on zlp.H + driven translocation, respectively. Both the polarity and the magnitude of zi/xH + affect S e c A / A T P - d e p e n d e n t preprotein translocation. zl/xH + increases the rate of proOmpA translocation by 2-5-fold in IMV [168,268,275]. Conversely, imposition of a zip.H + of reversed polarity blocks the overall S e c A / A T P - d e p e n d e n t p r o O m p A translocation in S e c Y / E proteoliposomes [255]. In addition, zi/xH + reduces the apparent K l / 2 of ATP for preprotein translocation by two orders of magnitude [275], leading to the suggestion that A/xH + may facilitate ADP release [275]. However, since zi/xH + can stimulate completion of an intermediate of translocation in the absence of SecA [77], such an explanation seems unlikely. ATP or zitxH + can independently stimulate the completion of translocation of kinetic intermediates arrested after translocation of 16 kDa (Ii6 [77]), 26 kDa (I26 [254]) or 29 kDa (I29 [77,254]) of proOmpA. However, these driving forces act competitively when imposed simultaneously at specific stages of preprotein translocation. The rate of completion of translocation of Ij~, driven by zi/xH +, is reduced in the presence of SecA and completely blocked with SecA and a non-lay-

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drolyzable ATP analog [77]. These results indicate that k/.tH + stimulation of translocation is more apparent when the preprotein is not bound to SecA, suggesting that SecA preprotein binding and AtxH + compete for the same step. In addition, in vitro proOmpF-Lpp translocation, shown to strictly require A/xH + [267], becomes independent of AIxH + when a high concentration of SecA (20-fold the normal level) is present [276]. Together, these results suggest the existence of a competition between S e c A / A T P - and AtxH+-depend ent translocation pathways, subsequent to the initiation of preprotein translocation. Completion of transiocation of the latter intermediates 126 and 129 driven by zl/xH +, is unaffected by non-hydrolyzable ATP analogs [254,270] indicating that after a certain length of preprotein is translocated, S e c A / A T P and zl/xH + no longer act competitively. A possible explanation is that at such a level of translocation (I26) , the overall interactions between SecA and the carboxy-terminus of the preprotein are weaker and therefore the A/xH+-de pendent pathway is favoured.

[175,176,278]. Antibodies directed against SecD block the release of preprotein on the periplasmic face of the membrane, consistent with a role for SecD in the completion of preprotein translocation [179]. SecD and SecF could actively drive preprotein translocation, similar to the role of Hsp60 and Hsp70 in mitochondrial protein translocation. Nevertheless, as will be discussed in a latter section, none of these proteins are required in the translocation process per se. It is also possible that there are unidentified proteins, located in the cytoplasm or in the periplasm, which contribute energetically to the translocation process. The idea of cytoplasmic and periplasmic factors affecting preprotein translocation is consistent with the 'Brownian ratchet' hypothesis [279] applied to preprotein translocation [280]. In this proposal, transmembrane chemical asymmetry produced by the presence of specific cytoplasmic or periplasmic proteins, ATP hydrolysis, or A/xH + would direct the thermal motion of the preprotein and consequently the net movement of preprotein translocation.

4.3. Other energy sources

4.4. E n v i r o n m e n t o f the preprotein during translocation

In addition to A/xH + and ATP hydrolysis, several results suggest the participation of other energy sources, driving partial translocation of the preprotein. Forward or reversed partial translocation of the polypeptide chain is observed in the absence of added energy sources, i.e., ATP or AtxH +. Removal of SecA by urea treatment or inactivation with anti-SecA sera, leads to a reversed translocation of the preprotein [77]. Specifically, after SecA inactivation, I26 is converted to I16' indicative of a reversed translocation of 10 kDa of the preprotein. On the other hand, a forward translocation of 3 kDa of an intermediate of p r o O m p A - D H F R occurs in the absence of any added energy source, concomitant with an unfolding of the D H F R domain [78]. Refolding of the preprotein portion translocated into the periplasm, could participate in driving translocation of the rest of the preprotein. Conversely, folding of the preprotein in the cytoplasm, in the absence of SecA, could drive the reversed translocation of the preprotein. Analogously, the relative energies of transfer of residues into and out of the membrane environment could promote translocation [277]. Alternatively, interaction of the translocated portion of a preprotein with periplasmic proteins could drive the rest of the preprotein through the membrane, thereby facilitating completion of translocation. Several proteins are known to be involved in the completion of preprotein translocation across the cytoplasmic membrane (see below). For example, leader peptidase cleaves the leader peptide from the mature part of the preprotein [278]. However, leader peptidase is not necessary for preprotein translocation in vivo or in vitro

Preproteins have large hydrophilic domains and a central question is the nature of the immediate environment of the preprotein while crossing the hydrophobic membrane. As discussed in previous sections, both integral membrane proteins (SecY, SecE) and acidic phospholipids (such as PG) have a crucial role in preprotein translocation. Preprotein translocation across membranes has been proposed to occur directly through phospholipids [281] or, at the other extreme, via an aqueous pore or channel [282]. Results consistent with both proposals have been reported, although direct evidence is lacking. An intermediate possibility is that preprotein translocation occurs through both phospholipid and proteinaceous environments. There is precedent for a phospholipid-mediated translocation pathway. Insertion of M13 procoat into liposomes occurs without the aid of any membrane proteins [283]. However, insertion of the small M13 procoat into the membrane may occur by a fundamentally different mechanism than s e c - d e p e n d e n t translocation. Leader peptides partition into the membrane bilayer, a thermodynamically favorable process [29,30] and a correlation between PG requirement for translocation and the composition of the leader sequence of proOmpF-Lpp has been established [35]. These data lead to the proposal that the leader sequence interacts with PG during the translocation process. An alteration of the membrane lipid permeability during translocation has been suggested to be caused by the mature domain of the preprotein perturbing the membrane structure during translocation [284]. The

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magnitude of A~, but not a p H , decreases in the presence of SecA, ATP and proOmpA, but not the leader peptide. A similar decrease in aqt magnitude is observed in the presence of proOmpA-BPTI indicating that the / l ~ dissipation occurs without net preprotein movement across the membrane. This effect is only seen in the presence of halide anions, leading to the suggestion that the membrane permeability to halide anions is increased during the preprotein translocation process. Moreover the magnitude of this effect on Aqs dissipation with different halide anions, follows the order of selectivity for anions permeability of a phospholipid membrane. It is unclear whether this effect on membrane permeability is due to the mature domain directly perturbing the lipid phase or an indirect effect of preprotein translocation. The results of electrophysiological studies [285], are consistent with the hypothesis of preproteins crossing the membrane through an aqueous protein pore. E. coli cytoplasmic membranes were fused to a planar lipid bilayer and large conductance aqueous channels (220 to 240 pS) were observed when preLamB leader peptide was added on the cytoplasmic side of the membrane and not the periplasmic side. A low concentration of leader peptide was used (0.2 nM) to limit perturbation of bilayer structure observed at higher concentration (100 raM) [30]. The authors suggest that leader peptides are acting as ligands which gate a protein conducting channel. However these channels have not been directly shown to conduct proteins. The close association of the preprotein mature domain of a translocation intermediate with SecY and SecA, was demonstrated using cross-linking with a sufhydryl-specific, radiolabeled, photoactivable 21 ,~ cross-linker, A P D P [211]. Radiolabeled A P D P is attached to a unique cysteine located in the proOmpA carboxy-terminal portion of a p r o O m p A - D H F R fusion protein. Translocation intermediates are generated by blocking translocation at the D H F R moiety [78]. Subsequent to generation of translocation intermediates, A P D P is photolysed resulting in cross-links to the components proximal to the preprotein. Under these conditions, the radiolabeled portion of the cross-linker is selectively transfered to both SecY and SecA. Moreover, translocation intermediates appear largely shielded from cross-linking to the lipids, suggesting that preprotein translocation occurs in a proteinaceous environment. It would be premature to claim that the preprotein traverses the membrane in either an entirely lipid or proteinaceous environment. However, based upon thermodynamic considerations, it appears likely that the leader sequence comes in contact with the lipid phase at some stage during the translocation process. Together with electrophysiological and crosslinking results, one is compelled to compromise and conclude

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that a portion of the preprotein is in an aqueous proteinaceous environment. If this were the case, it would necessitate preprotein translocation occurring at some stage at a protein lipid interface, or at least in contact with lipid.

5. Subsequent steps of transiocation to periplasm and outer membrane

Depending on the preprotein, its fate following translocation across the cytoplasmic membrane varies. While periplasmic proteins such as MBP, RBP, PhoA, and Bla must fold and become functional, outer membranes proteins such as OmpA, LamB, PhoE, and OmpF are targeted to and assemble in the outer membrane. Secretion across the outer membrane occurs in some gram negative bacteria via a homologous outer membrane secretion apparatus called the terminal branch of the general secretion pathway, typified by the secretion of pullulanase, pilin, exotoxin, IgA proteinase, pectinase, and cellulase (for reviews see [286289]). In E. coli some of the periplasmic and outer membrane proteins must also oligomerize or form the correct disulfide bridges in order to become functional. This section will briefly discuss the late stages in translocation across the cytoplasmic membrane, involving SecD and SecF, leader peptidase, proteins in the periplasm which facilitate folding, and the targeting and assembly of proteins into the outer membrane. 5.1. SecD and SecF

The secD locus was identified, as previously discussed, by two different genetic screens, selection for mutants defective in the export of preprotein-LacZ [152] and selection for mutants that derepress secA [160]. These screens identified 10 mutations which mapped to the secD locus and conferred pleiotropic secretion defects. In vivo secretion of prePhoA, preMBP, proOmpF, preRBP, proOmpA, and preLpp is defective in these mutant strains [152,153,290,291]. The secD locus has been cloned and shown to encode six potential open reading frames. Complementation analysis has shown that the two largest open reading frames define the secD locus and have been named secD and secF. These open reading frames form an operon predicted to encode hydrophobic proteins of 65 and 35 kDa, respectively, each with multiple membrane spanning domains and a large periplasmic domain. While the sequences of SecD and SecF showed no significant homologies to other proteins, comparison of the hydrophobic carboxy-terminus of these two proteins show significant similarity with 26% amino acid identity over 176 amino acids. Four lines of evidence suggest that SecD and SecF

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may function late in preprotein translocation. Both of these membrane proteins have large periplasmic domains raising the possibility that they are involved in latter steps of translocation across the cytoplasmic membrane [153]. Extensive searches for extragenic mutations (prl) which suppress leader sequence mutations have not identified mutations in secD or secF [158,240], suggesting that these proteins may function after leader sequence recognition. Furthermore, genetic studies involving Sec titration indicate that secD and secF function subsequent to leader sequence cleavage [193]. In vitro translocation studies have demonstrated that proteoliposomes containing purified SecY, SecE, and SecA are able to translocate proOmpA at a rate comparable to IMV's [175,176], suggesting that SecD and SecF are not required to translocate proOmpA across the cytoplasmic membrane. Together these studies suggest that the role of SecD and SecF in translocation is fundamentally different than that of SecA, SecY, and SecE. Consistent with this notion SecD and SecF are not essential for growth at 37°C [181]. Depletion of S e c D / F or a null mutation in these genes result in cs growth and protein secretion. In both cases the rate of proOmpA and preMBP secretion is reduced at 37°C and further slowed at 30°C. In addition, it appears that S e c D / F overexpression can increase the rate of preRBP processing in vivo [181]. These in vivo studies establish a role for SecD and SecF in preprotein secretion, yet do not differentiate between their direct and indirect involvement in preprotein translocation. Direct evidence for the involvement of SecD in preprotein secretion came with spheroplast protein secretion studies [179], and in vitro translocation studies [180]. Anti-SecD IgG's inhibit the secretion of OmpA and MBP, resulting in the accumulation of precursor and mature protein associated with the spheroplasts [179]. Proteinase susceptibility studies indicate that the mature MBP associated with the spherop[asts in the presence of anti-SecD is on the outer surface of the cytoplasmic membrane, suggesting that SecD may be involved in preprotein release [179]. Using IMV preparations derived from cells that were depleted of SecD and SecF, atzH+-dependent in vitro translocation of both preMBP and proOmpA requires SecD and SecF [180]. Furthermore, in the presence of a generated A/xH +, processing of preMBP to MBP, indicative of the leader sequence crossing the membrane is not affected by the depletion of SecD and SecF in IMV. ATP-dependent translocation of preMBP and proOmpA into IMV is unaffected by the lack of SecD and SecF. These results are consistent with a function for SecD and SecF in the latter stages of translocation across the cytoplasmic membrane. The depletion of SecD and SecF results in a decrease of the magnitude of the proton electrochemical gradient in cells and IMV. These studies raise the possibility that

SecD and SecF may exert their effect in preprotein translocation via the proton electrochemical gradient, perhaps by coupling this driving force to net chain movement. Direct demonstration of such a role for SecD and SecF will require the functional reconstitution of these two proteins. 5.2. Leader peptidase

In E. coli, two proteinases, signal peptidase I and II, residing in the cytoplasmic membrane, are responsible for cleaving the leader sequence of preproteins. Signal peptidase II processes the leader sequence exclusively from lipoproteins ([292,293]; for review see [294]), whereas signal peptidase I (refered to as leader peptidase) processes the leader sequence of other secretory proteins [278,295]. Leader peptidase has been purified to homogeneity [296,297], the gene encoding this protein cloned [298], sequenced [295] and put under a regulated promoter [278]. Leader peptidase has two membrane spanning domains with both the aminoterminus and a large carboxy-terminal domain in the periplasm [295,299]. The active site of leader peptidase is on the periplasmic side of the cytoplasmic membrane [295]. Leader peptidase is an essential protein [278,300,301] which is not required for translocation [175,176,271, 278,302]. Both in vivo [302] and in vitro [271] studies have demonstrated that preproteins with uncleavable leader sequences are translocated. Furthermore in vivo [278] and in vitro [175,176] translocation can occur in the absence of leader peptidase. The function of leader peptidase is to cleave preproteins from their leader sequence which otherwise anchors them to the periplasmic face of the cytoplasmic membrane [278]. Leader peptidase has a wide substrate specificity (for review see [303]) and as discussed previously, the sequences of the substrates (leader sequences) are not conserved. Sequence comparison analyses [304-306], in vivo processing studies [302,307-311], and in vitro processing studies [307,311-314] have in combination defined the essential features of the leader peptidase cleavage site (Fig. 1). Amino acids at positions - 1 and - 3 with respect to the cleavage site, contribute to processing specificity. Small amino acid residues, such as ala, gly, or ser, are required at position - 1 and small or aliphatic residues such as ala, gly, thr, val, or leu at position - 3 . In addition, a helix breaker residue between positions - 4 and - 6 appears to be required for processing. These studies suggest that shape and conformation around the leader sequence cleavage site are crucial for specificity. Outside of these sequence requirements, a pro residue at the + 1 position is deleterious to processing [271,309,315,316]. PreMBP with a pro residue at +1 appears to competitively inhibit leader peptidase activity in vivo, preventing the

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processing of other preproteins and inhibiting the cell growth [315]. E. coli leader peptidase is not inhibited by conventional proteinase inhibitors [317]. Consistently, sequence comparisons with other prokaryotic leader peptidases [318-320] and site directed mutagenesis studies [320-323] indicate that the conserved ionizable residues cysteine, histidine, aspartate, glutamate, arginine, and tyrosine are not required for activity. Leader peptidase catalytic activity requires a single conserved serine residue and a lysine residue [322,323], suggesting that they act as a nucleophile and as a general base, respectively. While these studies have elucidated the leader peptidase residues involved in the catalysis of cleavage of the leader sequence, which are carboxy-terminal to the second transmembrane domain, it is unknown whether cleavage occurs within the lipid bilayer or adjacent to it.

a motif found at the active sites of a number of disulfide oxido-reductases. The crystal structure of DsbA resembles thioredoxin [336] and biophysical studies have shown that DsbA is a strong oxidant with the reduced form much more stable than the oxidized form [337-339]. These studies suggest that DsbA can interact with unfolded proteins, yet this remains to be demonstrated. In addition, it has been suggested that oxidation of reduced periplasmic DsbA is carried out by DsbB, which in turn may be reoxidized by another membrane component or cytosolic component [328]. This disulfide bond formation pathway is an elegant solution to the requirement for correct disulfide bond formation of proteins in a compartment (the periplasm) which is permeable to small molecules typically used for oxidation.

5.3. Folding and disulfide bond formation

While the translocation of four outer membrane proteins (Table 1) across the cytoplasmic membrane has been well studied, much less is known about what happens to these proteins after crossing the cytoplasmic membrane. OmpF, PhoE, and Lamb exist as trimers in the outer membrane, whereas OmpA is a monomer in its native state. Subsequent to crossing the cytoplasmic membrane, outer membrane proteins must fold, in most cases oligomerize, and correctly insert into the membrane. In addition, these proteins must avoid aggregation and nonspecific interactions with both the cytoplasmic and outer membrane. It has been suggested that assembly in the outer membrane occurs via a periplasmic intermediate whose conformation dictates insertion into the outer membrane [340]. Alternately, proteins may be exported to the outer membrane through adhesion zones [340], sites in which the cytoplasmic and outer membranes are in contact (termed Bayer's patches) and this mode[ would imply that the proteins are not free in the periplasmic space prior to insertion in the outer membrane. Protein insertion into the outer membrane is a rapid event. OmpF inserts into the outer membrane, forming a functional trimeric porin with a half time of 20-30 s following cleavage of the leader sequence [341,342]. In contrast, L a m b insertion and trimerization is slower occuring on a time scale of 5 min. OmpA insertion in the outer membrane occurs approximately 1 min after crossing the cytoplasmic membrane [343]. Kinetic and mutational analyses of the insertion of outer membrane proteins indicate that the leader sequence does not dictate outer membrane localization. In the outer membrane, porins [344] and OmpA [345] are thought to exist as amphipathic /3-barrels with between 8 to 16 anti-parallel /3-strands. Deletion studies demonstrated that removal of the amino-terminus of PhoE [56] and of the carboxy-terminus of OmpA

Although chaperone mediated folding of preproteins in the cytosol is well established, little is known about the folding of preproteins subsequent to crossing the cytoplasmic membrane. In addition to chaperones, prolyl isomerases and disulfide isomerases can aid in protein folding. In E. coli, a periplasmic peptidyl-prolyl isomerase homologous to cyclophilin has been identified [324-326] and dsbA and dsbB have shown to be involved in disulfide bond formation in the periplasm [327-331]. While the in vivo role of a periplasmic peptidyl-prolyl isomerase in refolding exported proteins is unknown, this enzyme has been shown to catalyze the isomerization of a tetrapeptide substrate [325]. Genetic studies will be necessary to identify physiological substrates of this isomerase. In contrast, correct disulfide bond formation is critical for a number of periplasmic proteins such as PhoA. DsbA and dsbB were identified by screens for defects in the folding of periplasmic PhoA [329] or a LacZ fusion protein [327,328]. In addition dsbB was identified by mutations which confer sensitivity to D T T [330] and mutants that require cystine for flagella assembly [331]. DsbA and dsbB mutants are defective in disulfide bond formation in Bla, OmpA, and PhoA. Neither of these genes are essential [327,328,330] and a null mutation in dsbA does not affect the translocation of PhoA, OmpA, or Bla [327,329]. These results suggest that disulfide bond formation in the periplasm is not coupled to translocation across the cytoplasmic membrane. DsbA is a periplasmic protein [327,329], whereas DsbB is an integral membrane protein [328]. Purified DsbA catalyzes the formation of disulfide bonds on a number of protein substrates [327,332-335]. Both DsbA and DsbB contain the sequence Cys-X-X-Cys which is

5.4. Targeting and assembly into the outer membrane

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[346], parts of these proteins that are normally periplasmic, has no effect on membrane insertion. Alteration of the last putative membrane spanning domain in OmpF [347], OmpA [346,348], and PhoE [56] impairs insertion in the outer membrane. Although numerous studies have failed to elucidate a universal sorting signal for the outer membrane targeting in the mature protein, it is presumed that some aspect of the mature proteins directs proteins to the outer membrane. This characteristic of the mature domain may be its ability to oligomerize, its secondary or tertiary structure, or its ability to interact with outer membrane components such as lipopolysaccharide. It is unlikely that solely oligomerization targets proteins to the outer membrane and drives their insertion into this membrane. Temperature sensitive folding mutations have been isolated in L a m b which prevent trimer formation, yet have no effect on localization to the outer membrane [349]. In addition, OmpF is secreted from spheroplasts into the medium as a monomer and these monomers are able to insert into outer membrane preparations and trimerize [350]. Both PhoE [351] and OmpF [352] monomers synthesized in an in vitro translation system are able to insert into the outer membrane and trimerize. Furthermore, OmpA inserts into the outer membrane in the absence of observable oligomerization [353,354]. Together these studies suggest that oligomerization of outer membrane proteins is not obligatory involved in their outer membrane targeting and insertion. Unfolded OmpA and OmpF can fold and insert into lipid vesicles, suggesting that some intrinsic characteristic of the mature domain, such as its structure, contributes to outer membrane insertion. In vitro synthesized or spheroplast secreted OmpF monomers can insert and trimerize in lipopolysacharides [350,352]. Denatured OmpA can also be folded by dilution out of denaturant in the presence of detergent and subsequently will insert into preformed vesicles of phosphatidylcholine [354,355]. This insertion results in correctly folded OmpA, functional as a phage receptor and possessing the correct membrane orientation. Insertion of OmpA also requires a highly curved bilayer, presumably lipid packing defects in small vesicles promote insertion [354]. In vitro insertion of OmpF and OmpA into the outer membrane, requires unfolded or nascent proteins suggesting that some aspect of refolding promotes insertion. Lipopolysaccharide (LPS) is found only in the outer leaflet of the outer membrane of gram-negative bacteria. Consequently, LPS has been suggested to be involved in sorting and assembly of outer membrane proteins [356]. Mutants with defective LPS have reduced amounts of outer membrane proteins [357]. In vitro folding and insertion studies with OmpA have demonstrated that LPS is not required for either reac-

tion [355], whereas LPS facilitates the in vitro, trimerization of monomeric OmpF [350]. Conversely, defective LPS from a mutant strain does not promote trimerization [358]. These experiments suggest that LPS is involved in assembly of porin trimers but is most likely not required for outer membrane insertion. In vitro studies suggest that protein components in the outer membrane are not essential for insertion. Nevertheless, in vitro OmpF insertion and trimerization is relatively inefficient, with 50-60% trimerization observed in LPS after 3 h [350] and 70-80% trimerization of OmpF in outer membrane vesicles after 2 h. Insertion of folded OmpA into phosphatidylcholine vesicles occurs with a half time of 10 rain. For both of these outer membrane proteins in vitro insertion is one to two orders of magnitude slower than in vivo, suggesting that other proteins (periplasmic or outer membrane) may increase the rate and efficiency of insertion. The use of these in vitro insertion systems should enable the identification of protein components which promote outer membrane insertion. One can envision the existence of chaperones facilitating outer membrane insertion, preventing aggregation and binding to the cytoplasmic membrane. Extragenic suppressor mutations of OmpF mutants, defective in outer membrane assembly have been identified [359]. Such genetic studies should be extremely useful in elucidating protein components of outer membrane targeting and assembly pathways.

6. Perspectives The process of preprotein translocation across the cytoplasmic membrane of Escherichia coli requires protein and lipid components in combination with ATP and A ~ H +. We have discussed the specifics of preprotein translocation in an effort to present a cohesive picture of this process. In E. coli, preproteins are synthesized with amino-terminal leader sequence extensions which retard the folding of the mature domain and have a propensity to interact with membranes. In the cytoplasm, SecB stabilizes certain preproteins for subsequent translocation by blocking preprotein folding and aggregation. SecA, a peripheral membrane ATPase, and S e c Y / E , an integral membrane protein complex, are both required for translocation across the cytoplasmic membrane. Translocation across the membrane is driven by ATP binding and hydrolysis and by the proton electrochemical gradient. Upon crossing the cytoplasmic membrane, preproteins may interact with other membrane proteins such as leader peptidase, SecD, SecF, periplasmic proteins such as DsbA, and then proceed to their final destination. Preprotein translocation is particularly intriguing not only because hydrophilic preproteins are made to cross a hydropho-

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bic membrane barrier, but in addition because this process is specific for diverse preproteins lacking sequence identities. While biochemical studies have revealed a vast amount of information regarding the mechanism of preprotein translocation, the generality of these mechanistic conclusions needs to be confirmed. Translocation of number of preproteins should be examined to address this concern. For example, do all preproteins require S e c A / S e c Y / S e c E for translocation? It would be very informative to quantitatively investigate the energetic requirements of a number of different preproteins. In this manner, it may be possible to understand the relative contributions of A/xH + and ATP hydrolysis as driving forces for preprotein translocation. It is essential in mechanistic studies of preprotein translocation to examine this process in the presence of translocation competent preprotein at saturating concentration under conditions of multiple turnovers. By studying the quantitative translocation of a number of preproteins, it should also be possible to delineate the specificity of preprotein translocation. For example, if two preproteins are present, what dictates the specific translocation of one? Preprotein translocation is now amenable to a detailed kinetic analysis. In order to understand this process we will need to know the rate limiting steps of translocation of each preprotein with ATP or A/~H + driving chain movement. While quantitative analysis of such a complex process as translocation will not be trivial, the mechanistic insights gained will be worth the effort. Furthermore, biochemical studies on the large number of Sec-protein mutants should reveal insights into the function of these proteins. In addition to a number of specific questions raised in this review, several general questions remain with respect to preprotein translocation. Are the membrane proteins required for preprotein translocation the same for membrane protein insertion? This area of study has been largely ignored in comparison to preprotein translocation. Is preprotein secretion regulated during the cell cycle and is it directed to a specific location on the cytoplasmic membrane? As preprotein secretion in E. coli has become more defined, protein secretion in other prokaryotes is being examined. Is the process of preprotein secretion mechanistically conserved in other prokaryotes? The isolation of Sec-protein homologues in other prokaryotes, will no doubt provide insight into the evolution of membrane transport and allow examination of the function of conserved regions of various Sec-proteins.

Acknowledgments We would like to thank Bill Wickner and members of his laboratory for many stimulating discussions. We

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are indebted to John Joly, G6rard Leblanc, and Elizabeth Pradel for critical reading of the manuscript and helpful suggestions. R.A. was supported in part by a Damon Runyon-Walter Winchell Cancer Research Fund Fellowship (DRG-1106) and the MRC. M.B. was supported by the CNRS France.

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