Degradation of cytoplasmic substrates by FtsH, a membrane-anchored protease with many talents

Research in Microbiology 160 (2009) 652e659 www.elsevier.com/locate/resmic

Degradation of cytoplasmic substrates by FtsH, a membrane-anchored protease with many talents Franz Narberhaus*, Markus Obrist, Frank Fu¨hrer, Sina Langklotz Ruhr-Universita¨t Bochum, Lehrstuhl fu¨r Biologie der Mikroorganismen, Bochum, Germany Received 26 June 2009; accepted 17 August 2009 Available online 8 September 2009

Abstract Control of cellular processes by regulated proteolysis is conserved among all organisms. FtsH, the only membrane-anchored AAA protease in bacteria, fulfills a variety of regulatory functions. This review focuses on soluble FtsH substrates in Escherichia coli and in other bacteria and outlines emerging substrate recognition principles. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: FtsH; AAA proteins; Proteolysis; Quality control; RpoH; LpxC; LPS biosynthesis

1. Introduction Proteolysis of transcription factors and enzymes has emerged as an important regulatory mechanism both in prokaryotes and eukaryotes. Degradation of proteins in the bacterial cell is mainly carried out by energy-dependent proteases. In Escherichia coli several proteases have been described: ClpAP, ClpXP, HslUV, Lon and FtsH [21]. All of them belong to the AAA-family of proteins (ATPases associated with various cellular activities) as they have a special ATPase domain with conserved regions to unfold their targets. Processive degradation of substrates is carried out in the protease domain, resulting in the release of small peptides. For many years, the main function of proteases was believed to be protein quality control by removal of unfolded or damaged proteins. Another task is protein processing resulting in the activation of a precursor protein. Within the last ten years, however, it was discovered that an equally important role of

these proteases is the specific degradation of substrate proteins to rapidly adapt to environmental changes [32]. Proteolysis of regulatory proteins and key enzymes has been studied best in the Gram-negative model organism E. coli. Although deletion of other AAA proteases leads to severe pleiotropic phenotypes, the only essential protease in E. coli is FtsH. While other AAA proteases are located in the cytoplasm, FtsH is anchored in the inner membrane [75]. The basic structural features, enzymatic activities and functions of E. coli AAA proteases in general and FtsH in particular have already been reviewed [31,50]. However, the knowledge of FtsH substrates and their recognition motifs is still far from being complete. This review focuses on cytoplasmic substrates of FtsH in different bacterial species and the mechanisms of substrate recognition, which have evolved to cope with changing environmental conditions by regulated proteolysis. 2. The FtsH protease: structure and functions 2.1. Structural features of FtsH

* Corresponding author. Ruhr-Universita¨t Bochum, Lehrstuhl fu¨r Biologie der Mikroorganismen, Universita¨tsstrasse 150, NDEF 06/783, D-44780 Bochum, Germany. Tel.: þ49 234 322 3100; fax: þ49 234 321 4620. E-mail addresses: [email protected] (F. Narberhaus), mobrist@ med.unc.edu (M. Obrist), [email protected] (F. Fu¨hrer), [email protected] (S. Langklotz). 0923-2508/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2009.08.011

The E. coli FtsH monomer consists of 647 amino acids with a molecular mass of 71 kDa. As a typical AAA protease, FtsH forms a barrel-shaped oligomer (Figs. 1 and 2) [9]. The hexameric protease is anchored to the inner membrane by two

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Fig. 1. Structural features and degradation mechanisms of FtsH. (A) Indicates the overall structure of one subunit of the soluble part of FtsH from Thermus aquaticus (PDB entry 2DHR, [73], edited with the Swiss PDB-viewer [24]). The ATPase domain is shown in blue and the protease domain in green. As special features, the Walker A/B motifs, the second region of homology (SRH), the pore region, the zinc binding motif HEXXH and the helices at the C-terminal end are depicted in black. (B) The hexameric structure of FtsH is shown using the same colors as in (A) to discriminate between ATPase and the protease domain. Two subunits are highlighted in dark blue and green, respectively. LpxC is given as an example of a degradation mechanism requiring a free terminus. As the structure of E. coli LpxC has not been solved yet, P. aeruginosa LpxC (PDB entry 2VES, [53]) without its C-terminus is shown on the left. The sequence- and lengthspecific E. coli C-terminal degradation signal, which is shown in the single letter amino acid code, was added. It is thought to be flexible and long enough to span the distance to the central pore. On the right, RpoH is shown as an example for a complex degradation mechanism. Since the structure of RpoH is not yet solved, only regions 2.1eC are given as a model [55]. Important residues in regions 2.1 and C are colored in red.

transmembrane helices per monomer such that both termini protrude into the cytoplasm. While only seven N-terminal amino acids extend into the cytoplasm, w520 residues of the C-terminus comprise the cytoplasmic ATPase and protease domain [76]. The ATPase domain contains the Walker A/B motifs and the second region of homology (SRH) which are characteristic

of AAA proteins and responsible for the binding and hydrolysis of ATP [59]. The ATPase domain forms the entrance to ˚. A the central pore of FtsH, with a diameter of about 15 A conserved phenylalanine at position 228 of the pore is crucial for substrate binding. Substrates are pulled through the narrow gate and into the following protease domain using the energy

Fig. 2. Cellular functions of the FtsH protease in E. coli (adapted from [46]). FtsH forms a hexameric structure integrated into the inner membrane. For clarity, the HflKC proteins, which form a supercomplex with FtsH [12,65], were omitted. The protease is involved in quality control of membrane proteins by either refolding or degradation of misfolded or unassembled membrane proteins. FtsH has a number of soluble substrates and is involved in removal of imperfectly synthesized proteins, and regulates the lytic/lysogenic decision of phage lambda. The protease regulates the superoxide stress response as well as heat shock gene expression and controls the biosynthesis of membrane components. IM: inner membrane; OM: outer membrane; LPS: lipopolysaccharides.

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Table 1 Cytoplasmic substrates of the FtsH protease in bacteria. If analyzed, adaptor or modulator proteins and localization of degradation signal are given. ND: not determined Protein

Organism

Ref.

Adaptor/modulator proteins; localization of degradation signal

SsrA-tag lCII lCIII lXis SoxS Flavodoxin LpxC KdtA RpoH (s32) RpoH (s32) sF sW SpoVM Spo0E GgpS GlnK

E. coli Phage l/E. coli Phage l/E. coli Phage l/E. coli E. coli E. coli E. coli E. coli E. coli C. crescentus C. crescentus B. subtilis B. subtilis B. subtilis Synechocystis sp. PCC 6803 C. glutamicum

[27] [38,68] [29] [48] [23] [62] [58] [35] [28] [17] [4] [80] [14] [47] [70] [72]

The tag itself HflD, HflK/C; C-terminus Internal ND N-terminus (Lon) Internal C-terminus ND DnaK/J, GroEL/ES; internal ND ND ND Internal C-terminus ND ND

of ATP hydrolysis for unfolding and translocation of the proteins [9,61,78]. FtsH was shown to have only weak unfolding activity, as FtsH is not able to degrade tightly folded model substrate proteins like GFP or DHFR. The folding state of a protein might thus be a critical feature that FtsH uses to recognize potential substrates [26]. FtsH is a metalloprotease and the protease domain contains the zinc-coordinating HEXXH motif. The histidine residues form two out of three zinc ligands [75]. Derived from structural studies on Thermotoga maritima, FtsH, the third ligand was identified as being a conserved aspartic acid at position 495. Therefore, FtsH is referred to as a member of the subclass of zinc-metalloproteases called ‘‘Asp-zincins’’ [9]. Another structural feature of FtsH is a C-terminal region in the protease domain consisting of three helices. By sequence comparison, this region was predicted to be a coiled-coil region, but turned out to be an extended helix in the crystal structure (Fig. 1) [9,69]. Three conserved leucine residues at positions 567, 574 and 584 in the first two helices form a leucine zipper which is crucial for degradation of RpoH and lCII. This region is therefore thought to mediate substrate binding [69]. FtsH is known to form a large membrane-spanning holoenzyme of more than 1000 kDa with the prohibitin-like proteins HflK and HflC [65]. HflK and HflC trigger activity against SecY and lCII [37,38]. QmcA, an additional prohibitin-like protein with opposite orientation compared to HflK/C, was also shown to interact with FtsH [12]. Modulation of proteolysis by prohibitins is a conserved mechanism, as prohibitins Phb1p and Phb2p trigger the activity of the m-AAA protease in mitochondria [see review by T. Langer in this issue]. FtsH is essential in many but not all Gram-negative bacteria. Although the ftsH gene is not essential in Grampositive bacteria, deletion mutants lead to severe pleiotropic effects. There is a correlation between the presence of FtsH and stress resistance. The paragraphs which follow will give an overview of already confirmed substrates of bacterial FtsH, reflecting its importance in different species.

Ref.

[41,42] [29] [23,67] [62] [18,19] [30,55e57]

[14] [47]

2.2. Membrane substrates of FtsH The FtsH protease controls diverse cellular processes in E. coli (Fig. 2). FtsH is involved in the quality control of misfolded or incorrectly inserted membrane proteins and acts either as a chaperone to help them refold or degrades them. Uncomplexed membrane proteins like subunit SecY of the SecAEG translocase or F0a of the Hþ-ATPase are degraded by FtsH when not associated with their partner proteins [2,3]. Another membrane substrate of FtsH is YccA, a protein of unknown function which seems to be linked to biofilm formation [7,39]. For detailed information on membrane substrates, we refer to recently published review articles [1,34]. 2.3. Cytoplasmic substrates of FtsH The majority of cytoplasmic substrates of FtsH were discovered in E. coli (Fig. 1, Table 1). Among other proteases, FtsH participates in the degradation of SsrA-tagged proteins. To enable ribosome recycling and removal of aberrant proteins from the cell, the SsrA-tag is added to stalled nascent chains during translation. It is estimated, that 0.5% of the translation products receive an SsrA-tag [51]. The SsrA-tag consists of 11 residues and is a general degradation signal for ClpAP/XP, Lon and FtsH [13,22,27] [see minireview by Pruteanu and Baker in this issue]. Apart from its role in protein quality control, FtsH has many regulatory functions, e.g. in the lysis/lysogeny decision of phage l. Historically, the ftsH gene was discovered independently through three different phenotypes and hence received different designations: ftsH, for filamentous temperature-sensitive [66]; tolZ, for colicin tolerance [52] and hlfB, because E. coli ftsH mutants show a high frequency of lysogenization when infected with phage lambda [6]. Degradation of the phage protein lCII by FtsH establishes the lytic pathway [68]. The protein lCIII acts as a competitive inhibitor of FtsH, as it is degraded slowly, leading to stabilization of

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lCII [29], which in turn induces the expression of repressor protein lCI. The expression of lCI causes entry into the lysogenic pathway. Another phage l protein, lXis, is also degraded by the FtsH protease and is responsible for excision of phage DNA from the bacterial genome [48]. FtsH also influences the shut-off of the oxidative stress response. Under oxidative stress conditions, the response regulator SoxS is expressed but is rapidly degraded with a halflife of 1 min by both FtsH and Lon to ensure recovery to basal levels when superoxide concentrations are rebalanced [23]. In search for a spectrophotometrically monitorable model substrate for FtsH degradation, apo-flavodoxin of E. coli was found to be degraded in vitro. The holo-flavodoxin with the bound flavin mononucleotide is not accessible for degradation. The impact of FtsH on flavodoxin levels in vivo remains unknown [60,62]. Its role in degradation of LpxC, the key enzyme in lipid A biosynthesis, renders FtsH essential in E. coli and probably in other Gram-negative bacteria. Lipid A is the hydrophobic anchor of lipopolysaccharides (LPS), which form the outer leaflet of the outer membrane of Gram-negative bacteria. Hydroxymyristoyl-ACP, the precursor of lipid A biosynthesis, is also used for phospholipid biosynthesis, allowing for balanced production of LPS and phospholipids. Both too much and too little lipid A is toxic for E. coli, making regulation of LpxC levels by proteolysis crucial for cell viability [58]. Interestingly, FtsH plays a dual role in LPS biosynthesis, since KdtA, a KDO transferase responsible for KDO attachment to lipid A, is also a FtsH substrate [35]. Due to its role in LPS biosynthesis, FtsH is the only AAA protease with critical functions in protein and membrane lipid homeostasis. Another important function of FtsH in E. coli is control of heat shock gene expression. Under heat shock conditions, the cell is confronted with aggregated and unfolded proteins. To counteract the accumulation of inactive proteins, expression of heat shock proteins is induced by the alternative sigma factor RpoH (s32). The heat shock regulon comprises molecular chaperones such as DnaK/J, GroEL/ES, ClpB and IbpA/B as well as proteases like ClpXP, HslUV, Lon and FtsH [54]. RpoH levels are adjusted to the physiological demand by a complex regulatory network involving transcriptional, translational and posttranslational mechanisms, including degradation of the sigma factor by the FtsH protease. FtsH-mediated control of cellular processes is not restricted to E. coli (Table 1). Regulation of heat shock gene expression by FtsH-dependent degradation of RpoH also occurs in Caulobacter crescentus, although the degradation mechanism seems to be different, as it does not require DnaKJ [15,17]. Proteolytic regulation of alternative sigma factors turns out to be a common mechanism. The cellular level of the C. crescentus extracytoplasmic function (ECF) sigma factor SigF (sF), which mediates the oxidative stress response in stationary phase, is adjusted by FtsH [4]. The ECF sigma factor sW of Bacillus subtilis might be another FtsH substrate [80]. The FtsH protease of B. subtilis is also involved in endospore development, a process that is coordinated by six sigma factors and a complex protein network [45]. FtsH

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participates in early and late stages of sporulation by degrading Spo0E and SpoVM [14,47]. Spo0E is a phosphatase that is needed to regulate the phosphorelay of Spo0A, the sporulation master regulator. SpoVM is assumed to be an inhibitor of the FtsH protease, as it is degraded with slow kinetics. The target that is stabilized through this inhibition has not yet been identified [14]. FtsH has been assigned a role in nitrogen metabolism in Corynebacterium glutamicum as it degrades the GlnK protein. Under nitrogen starvation, GlnK is adenylated and interacts with the global repressor protein AmtR to permit expression of nitrogen starvation genes. Upshift to high nitrogen concentrations sequesters GlnK to the cytosolic membrane, where it interacts with the transporter AmtB to block ammonium uptake. Complexed GlnK is sequentially degraded by the FtsH and Clp proteases [72]. In contrast to heterotrophic bacteria, which possess only a single ftsH gene, cyanobacteria encode four copies. In Synechocystis sp. PCC 6803, FtsH2 coordinates osmoregulation by degradation of cytoplasmic glycosyl glycerol (GG) synthase GgpS [70]. GgpS and GG phosphate phosphatase GgpP form a complex to catalyze GG synthesis. Uncomplexed GgpS is thought to be accessible for degradation by FtsH2. 3. How does FtsH recognize structurally diverse substrates? It is puzzling that most of the known substrates of the membrane-anchored FtsH protease are cytoplasmic proteins. This immediately raises the question of how soluble proteins are recognized, bound, translocated and degraded by FtsH. Thus far, two principally different pathways have emerged. The first route relies on recognition of free N- or C-terminal ends. The second mechanism is more complex, and includes structural features of the client protein. In both pathways, specialized adaptor and targeting proteins might be involved. 3.1. Hang loose: free ends as recognition motifs FtsH is able to specifically degrade membrane proteins like SecY and F0a when they are not associated with their partner proteins. For all membrane substrates described thus far, an unstructured and flexible N- or C-terminal tail that protrudes into the cytoplasm is mandatory to initiate proteolysis by FtsH. No specific sequence, but rather a minimal length, serves as a signature for non-native conformation of a membrane protein [11,39,40]. The minimal length of an exposed N-terminus is around 20 amino acids, while only ten C-terminally-exposed amino acids are sufficient for degradation. Although recognition of free ends is a common principle, the molecular basis of target interaction and membrane dislocation might differ from case to case [11]. Recognition of terminal degradation signals is also typical for soluble FtsH substrates, including SsrA-tagged polypeptides (Table 1). The SsrA-tag is located at the C-terminal end of the target [36]. The sequence is mainly composed of nonpolar amino acids (AANDENYALAA) inducing degradation

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by ClpXP, ClpAP and to a minor extent, by Lon and FtsH [13,22,27]. The adaptor protein SspB is responsible for targeting tagged substrates mainly to the ClpXP machinery [49]. Non-polar residues were repeatedly found in regions important for FtsH-dependent proteolysis, as is also the case for the C-terminus of l protein CII [42]. It contains a sequencespecific, structurally flexible, and therefore accessible degradation signal [16]. Proteolysis of CII involves adaptor protein HflD. Lambda CIII, the inhibitor of lCII degradation, is a small peptide consisting of 54 amino acids. Different recognition principles apply to the two proteins. The C-terminus of lCII is important for degradation, while amino acids important for the inhibitory effect of lCIII are found inside the protein. These residues center in an alpha-helix formed by amino acids 16e37 [44]. lCIII probably blocks the interaction between FtsH and HflD and does not mimic the degradation mechanism of lCII. SpoVM of B. subtilis also is an FtsH inhibitor and consists of only 26 residues. SpoVM shares structural similarities with lCIII, indicating that both polypeptides share the same inhibition and degradation mechanism [43]. An unstructured C-terminus is also postulated for LpxC, the substrate responsible for the essentiality of FtsH in E. coli. KdtA, another LPS biosynthesis enzyme, is also regulated by FtsH-dependent proteolysis. However, it appears to be degraded via a different, not yet elucidated pathway [35]. LpxC carries a C-terminal degradation sequence that is largely non-polar and resembles the SsrA-tag (Fig. 2B). The final 11 residues (-LAFKAPSAVLA) constitute the degradation motif, in which the first two (LA) and last four (AVLA) amino acids are crucial for proteolysis. In addition to sequence determinants, a critical length of at least 20 amino acids of the C-terminal end is required for degradation. Removal of the tail or exchange of at least two residues against the polar aspartic acid stabilizes the protein without affecting its activity [18,19]. This indicates that the sole function of the C-terminus is to target LpxC to FtsH, as also shown for lCII [42]. Consistent with its similarity to the SsrA-tag, the C-terminus of LpxC is a general degradation sequence that, if added to GST, leads to degradation of the fusion protein not only by FtsH but also by other proteases. Therefore, an additional motif is thought to confer strict FtsH specificity to LpxC. This second region was narrowed down to the N-terminus and could either be a specific amino acid sequence or a structural element. This motif might initiate binding to FtsH itself or to a yet unknown adaptor protein [19]. Involvement of DnaKJ, which is required for RpoH degradation (see below), in proteolysis of LpxC was excluded [18]. The inhibitor of FtsH, lCIII, was shown to block degradation of lCII which is normally bound by the C-terminal region of FtsH (Fig. 2A) [43,69]. Overexpression of lCIII also inhibits degradation of LpxC and RpoH [5,19]. Therefore, it is possible that LpxC is directly or indirectly bound to the C-terminal region of FtsH. Upon recruitment to the FtsH protease, the flexible tail of LpxC might span the distance to the central pore between the ATPase subunit of FtsH and the cytoplasmic membrane. The critical length of the C-terminus of LpxC is in good agreement with the suggestion that 20

amino acids are needed to bridge the distance between the extended helix in the C-terminus and the central pore of FtsH [31]. The recognition at the central pore might then be mediated by the interaction between the non-polar residues at the C-terminal end of LpxC and conserved amino acids at the entrance channel of FtsH [78]. Other protein tails that serve as recognition sites for proteolysis are less well characterized. The 85-amino acid protein Spo0E from B. subtilis carries a C-terminal degradation signal that, if transferred to some other protein, makes it susceptible to FtsH [47]. An N-terminal degradation sequence confers instability against the Lon protease to SoxS, the E. coli regulator of superoxide stress. Whether this particular sequence is also needed for degradation by FtsH is unknown [67]. 3.2. Complex substrate recognition mechanisms Several FtsH substrates are not recognized by the presence of free ends. In the case of E. coli flavodoxin, for example, blocking of the two termini by GST and GFP did not stabilize the protein in vitro, suggesting that a degradation motif inside the protein is needed for proteolysis [60]. The best-studied FtsH substrate of this class is the alternative heat shock sigma factor RpoH (s32). Although expressed at physiological temperatures, RpoH is rapidly degraded with a half-life of about 1 min mainly by FtsH, but also by other proteases [28,33,75]. The steady-state level of RpoH is adjusted to about 50 molecules per cell under physiological growth conditions [71]. This basal concentration of RpoH molecules enables a quick response to a sudden temperature increase cumulating in induction of multiple genes belonging to the RpoH regulon [54]. Interaction of DnaK and its co-chaperone DnaJ with RpoH is known to inactivate and target the sigma factor to FtsH [20]. The GroESL chaperones are also involved in RpoH degradation [25]. The fact that RpoH is degraded by FtsH in vitro without the help of chaperones e albeit with lower kinetics than in vivo [10,77] e suggests that the chaperones enhance substrate recognition and probably unfolding to increase degradation efficiency. Under heat shock conditions, the chaperones are engaged in refolding abnormal proteins. As a consequence, they are titrated away from RpoH, which is then able to bind to RNA polymerase in order to initiate transcription of heat shock genes. The stabilization of the sigma factor together with enhanced translation of its gene increases the amount of RpoH to about 1000 molecules. Shut-off of the heat shock response is triggered about 4e6 min after the stress and the amount of RpoH is reduced to lower levels [71]. Neither end of RpoH but an internal region within the N-terminus is required for proteolysis [8,74]. Amino acids in region 2.1 of RpoH are important for FtsH-dependent proteolysis [30,55,57,79]. Amino acid substitutions at positions L47, A50 or I54 were found to protect RpoH from degradation. Modeling of the relevant RpoH region on the basis of solved sigma factor structures suggests that all three amino acids line up on one face of an a-helix (Fig. 2B). Since RpoH

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variants with mutations in region 2.1 were not completely stabilized, this region appears to be necessary, but not sufficient, for FtsH-dependent degradation. Another region important for degradation of RpoH was found to be located in region C. The substitution of residues A131 and K134 led to stabilization of RpoH and a minimal RpoH fragment consisting only of regions 2.1 and C was sufficient for degradation by the FtsH protease [55]. Since neither mutation abolished sigma factor activity of the full-length protein, these residues supposedly do not interact with the transcription machinery. Chaperone binding was not altered either. According to structural models, these residues are roughly oriented in the same direction as L47, A50 and I54 (Fig. 2B). RpoH is a very flexible protein susceptible to temperaturedependent conformational changes which probably influence association and dissociation with its interaction partners [63]. Separate DnaJ and DnaK binding sites within RpoH were identified by amide hydrogen exchange experiments, mass spectrometry and gel filtration [64]. DnaJ was shown to bind residues 57e66, thereby introducing a conformational change in residues 91e101 and 158e175. This local opening facilitates binding by DnaK around residue 200. In turn, DnaK binding induces a second conformational change in RpoH, which destabilizes residues 31e49. This sequential action of the DnaKJ chaperone system renders the heat shock sigma factor, a correctly folded protein, susceptible to recognition and degradation by the FtsH protease [for a similar mechanism that involves the targeting factor RssB in regulated proteolysis of the general sigma factor RpoS, see minireview by R. Hengge in this issue]. 4. Concluding remarks FtsH is a versatile protease that degrades a number of structurally and functionally diverse substrates residing in the cytoplasmic membrane or the cytoplasm. For a selected set of proteins, FtsH may also act as a chaperone. Altogether, more than a dozen FtsH substrates have already been described in various bacteria. The physiological importance of FtsH both in Gram-negative and Gram-positive bacteria might imply that numerous substrates remain to be discovered. Astoundingly diverse degradation pathways apply for the few substrates that have been studied in detail. Although a picture on substrate recognition is emerging for some of these proteins, a number of questions remain to be addressed. Does FtsH have as yet undiscovered substrates? How can FtsH, which is a weak unfoldase, dislocate membrane-integrated proteins? How are cytoplasmic substrates captured by FtsH and how do they enter the protease from the membrane side? Does FtsH bind its substrates directly, or are prohibitins or adaptor proteins involved in this process? How are they translocated to the inner chamber of the ATPase domain once they are bound to the surface of protease? It is clear that entry into the central compartment is only possible from the membrane-oriented site of the protease. Is there any directionality in substrate translocation and degradation? In this context, it is intriguing that both N- and C-terminal ends can

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act as recognition tags. The answers to all these questions might vary for individual substrates. Apparently, much more research is required to unravel the mechanistic details of substrate selection and degradation by the FtsH protease.

Acknowledgements We gratefully acknowledge financial support by the German Research Foundation (DFG, priority program SPP 1132).

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