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Chapter 9 Molecular genetic analysis of membrane protein topology
CHAPTER 9
Molecular Genetic Analysis of Membrane Protein Topology M. LEE and C. MANOIL Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195, USA
9 1996 Elsevier Science B. V. All rights reserved
Handbook of Biological Physics Volume 2, edited by W.N. Konings, H.R. Kaback and J.S. Lolkema
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Contents
1.
Introduction
2.
Construction of topology models based on amino acid sequences
3.
Assay of membrane protean topology using gene fusions . . . . . . . . . . . . . . . . . . . . .
4.
5.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................
3.1.
Generation of gene fusions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Assay of hybrid protein activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Evaluation of models
3.4.
Anomalous behavior of gene fusions in topology studies
3.5.
Alkaline phosphatase sandwich fusions
3.6.
Analysis of mammalian membrane proteins in E. coli . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................
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Analysis of membrane protein topology using inserted sites
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192 193 193 193 195 195 196 197
4.1.
Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
197
4.2.
Antibody binding
198 199
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Vectorial labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Endogenous modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
199
4.5.
General considerations
200
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations
200
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
:200
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
A fundamental aspect of integral membrane protein structure is the transmembrane topology of the polypeptide chain, i.e., the disposition of the chain relative to the lipid bilayer in which it is embedded. A variety of biochemical techniques have been employed to elucidate topological structure, including proteolysis, side chain covalent modification, and epitope recognition [ 1]. However, these techniques often are unable to provide a full topological description of a membrane protein because of their reliance on the natural occurrence of residues that are susceptible to proteolytic cleavage, have reactive side chains or are accessible to antibody binding. These limitations have been largely circumvented by the development of molecular genetic methods which can be used in conjunction with the more traditional approaches. Two molecular genetic strategies for analyzing membrane protein topology have been developed. The first relies on the properties of gene fusion-encoded hybrid proteins in which enzymes whose activities reflect subcellular localization are attached at different sites in a membrane protein. The second strategy depends on the introduction of sequences (e.g., protease cleavage sites) whose subcellular disposition can be easily assayed. In this chapter, we will review both approaches, with special reference to work that has analyzed Escherichia coli lactose permease as a model. The reader is also referred to several excellent earlier reviews [1-3].
2. Construction of topology models based on amino acid sequences
The first step in analyzing the topology of a membrane protein is usually the formulation of a model or models based on the amino acid sequence of the protein. The construction of such models is generally based on two assumptions. The first assumption is that membrane-spanning sequences correspond to apolar cx-helical sequences that are sufficiently long to span the bilayer. The second assumption is that the spanning sequences are oriented in accordance with the 'positive inside' rule [4], that is, the favored orientation maximizes the exposure of positive residues in hydrophilic loops facing the cytoplasmic side of the membrane. The major difficulties encountered in formulating topology models using these rules are: (1) some spanning sequences contain polar residues which can make their identification problematic; (2) large extramembranous domains do not follow the positive inside rule [5], and (3) non-helical spanning sequences, such as 13-strands and sequences which insert into the membrane without spanning it, may contribute to a protein's membrane association [6-9]. Nevertheless, most of the integral 191
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membrane protein structures that have been determined are in reasonable accord with these general rules [7,10,11 ].
3. Assay of membrane protein topology using gene fusions The logic of using gene fusions to analyze membrane protein topology is illustrated in Fig. 1 [12]. Fusions of a gene encoding a protein whose enzymatic activity reflects its subcellular location (e.g., E. c o l i alkaline phosphatase (AP) lacking its signal sequence) are generated at multiple sites in the gene for a membrane protein, and the activities of the resulting hybrid proteins are compared to the previously formulated models. In principle, the activity of each hybrid protein reveals whether the junction site normally faces toward or away from the cytoplasm. A number of different proteins have been used as subcellular location 'sensors' in this type of analysis. In bacteria, the most commonly used sensor proteins are alkaline phosphatase and 13-1actamase (which require export to the periplasm for activity) and [3-galactosidase (which requires cytoplasmic localization for maximal activity) [2]. The combined use of periplasmic-active and cytoplasmic-active fusions provides positive activity signals for domains on both sides of the membrane, allowing a particularly thorough analysis (e.g., see Ref. [13]). A gene fusion analysis of topology generally consists of three steps: (1) the generation of gene fusions, (2) the assay of hybrid protein enzymatic activity, (3) a comparison of results to topology models.
1
IJ U1111 Ut
MEMBRANE PROTEIN
,,
ACTIVE ALKALINE PHOSPHATASE HYBRID
INACTIVE ALKALINE PHOSPHATASE HYBRID
Fig. 1. Analysis of membrane protein topology using alkaline phosphatase gene fusions. The top panel represents a membrane protein with six membrane-spanning segments. Gene fusions with fusion junctions corresponding to periplasmic positions (e.g., position 1) form hybrids with an exported alkaline phosphatase (AP) attached to N-terminal membrane protein sequences (bottom, left) which exhibit AP activity. Fusions at cytoplasmic positions (e.g., position 2) generate a cytoplasmically disposed AP which is inactive. Cytoplasmic AP appears to be inactive because it cannot form disulfide bonds necessary for folding [47].
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3.1. Generation of gene fusions Two methods have been commonly used for the generation of gene fusions: the insertion of transposon derivatives that 'automatically' generate the fusions, and in vitro mutagenesis using oligonucleotide-based methods. Both approaches employ membrane protein genes carried on plasmids. The two methods have different advantages and limitations. The generation of fusions by transposition is technically simple but suffers from the disadvantage that the junction sites are determined by the specificity of transposon insertion [ 14]. Although this difficulty is minimized for transposon derivatives with a relatively low insertion specificity (e.g., derivatives based on Tn5) [15], it may still be difficult to identify fusions with junctions in small extramembranous domains, which are common in transport proteins. The generation of fusions with predefined junctions using site-directed mutagenesis [16] or Polymerase Chain Reaction-based methods [17] is more laborious than generating the fusions by transposition, but has the advantage that junction positions can be specified by the oligonucleotides employed. This advantage is especially useful in distinguishing between closely related models for a structure (e.g., see Ref. [18]). Additional methods for generating fusions based on the interconversion of different fusion types ('fusion switching') [19] and the generation of nested deletions in appropriate plasmid constructs [20] have also been developed. The fusion switching technique makes it possible to compare the activities of hybrid proteins with different sensor proteins attached at exactly the same site in the membrane protein. The junction sites of fusions generated using any of these methods can be identified simply by DNA sequence analysis using oligonucleotide primers complementary to sequences encoding the 'sensor' proteins [21 ].
3.2. Assay of hybrid protein activity The enzyme activities of hybrid proteins are typically determined by assay of permeabilized cells using chromogenic substrates (alkaline phosphatase or I]-galactosidase) [21 ] or by means of a bioassay (I]-lactamase) [22]. Since gene fusions with different junction positions may be expressed at different levels, it is essential that rates of hybrid protein synthesis are examined to correct for such differences [23].
3.3. Evaluation of models The final step in a topology analysis is a comparison of results with models predicted from the sequence. As an example, the results of an AP gene fusion analysis of E. coli lactose permease are shown (Fig. 2). The fusions were generated by a combination of transposon TnphoA insertion and in vitro mutagenesis [ 18,24]. The activities of most fusions are consistent with the topology model, with periplasmic fusions expressing high alkaline phosphatase activities and cytoplasmic fusions expressing lower activities. Many of the lactose permease-alkaline phosphatase fusion junctions fall into the putative membrane-spanning sequences. A study of fusions with junctions within spanning sequences oriented with their N-termini facing the cytoplasm
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PERI
MEM
CYT
Fig. 2. Alkalinephosphatase gene fusion analysis of lactose permease. The positions and activities of AP gene fusions are shown [ 18,24] on the twelve-span model for the topologyof lactose permease. Highly active fusions are represented as filled arrowheads, partially active fusions as cross-hatched arrowheads, and inactive fusions as open arrowheads. PERI, periplasm; MEM, membrane; CYT, cytoplasm. (spanning sequences 3, 5, 7 and 11) indicates that fusions carrying more than 9-1 1 residues of the spanning sequence express maximal AP activities. It thus appears that only about half of a spanning sequence suffices for the protein export function of a membrane-spanning sequence. This conclusion is consistent with a variety of other genetic studies of protein export signals [25]. Fusions with junctions in spanning sequences in the opposite orientation (N end periplasmic) tend to show elevated alkaline phosphatase activities unless they contain not only the entire apolar sequence but C-terminal charged residues as well. This requirement evidently accounts for the partial AP activities of the fusions in cytoplasmic segment 3 (indicated by cross-hatched arrowheads). Detailed studies of analogous alkaline phosphatase fusions to MalF protein first implied a special role for positively charged residues in the cytoplasmic localization of such domains [26]. An amphipathic sequence plays an apparently analogous role in the E. c o l i serine chemoreceptor [27]. Two other classes of anomalous fusions have been defined and are discussed below (Section 3.4). The overall success of gene fusion methods in analyzing membrane protein topology implies that the subcellular localization of a site in a membrane protein does not, in general, require C-terminal sequences. The information dictating a
Membrane protein topology
195
particular topology must therefore be present in sequences N-terminal to a given site or redundantly determined by N-terminal and C-terminal sequences. In addition, the folding defects caused by the loss of C-terminal membrane protein sequences in hybrid proteins evidently does not usually prevent the remaining N-terminal sequences from adopting the wild-type topology.
3.4. Anomalous behavior in topology studies Three types of anomalous behavior have been noted in topology studies involving alkaline phosphatase fusions. As mentioned in the previous section, alkaline phosphatase fusions with junctions near the N-terminal end of a cytoplasmic loop in a membrane protein can exhibit anomalously high activity. The unexpectedly high activity of such fusions may be due to loss of C-terminal hydrophilic sequences such as positive residues required to orient the preceding transmembrane sequence. A recent study of one such fusion site in MalF indicated that 13-1actamase fusions may be less prone to this type of anomaly than AP fusions [28]. A second anomaly occasionally occurs when periplasmic domain AP fusions exhibit unusually low activities (e.g., the fusion to the fifth periplasmic domain of lactose permease) (Fig. 2) [ 18,29]. These cases involve fusions with junction points C-terminal to spanning sequences carrying charged residues. The charged residues reduce the overall hydrophobicity of the spanning sequence and may decrease the sequence' s ability to promote export. In such cases, C-terminal spanning sequences in the unfused protein may be required for efficient export of the hydrophilic domain in question. Since such charged residues in spanning sequences are often important in the biological function of membrane proteins, anomalously low alkaline phosphatase activities may commonly be observed for fusions with junctions in regions of functional importance (e.g., regions in a transport protein involved in binding hydrophilic substrates during passage across the membrane). A third anomaly in gene fusion analysis of topology is encountered for membrane proteins whose (unprocessed) N-termini face the periplasm [13,30]. Hybrid proteins with junctions in these N-terminal domains lack transmembrane sequences and are, in general, not exported.
3.5. Alkaline phosphatase sandwich fusions The anomalies in gene fusion analysis of membrane protein topology are thought to arise at sites where C-terminal sequences are required for normal subcellular localization. In an attempt to circumvent this problem, a novel type of alkaline phosphatase fusion ('sandwich fusion') was developed in which the alkaline phosphatase sequence is inserted into the membrane protein sequence rather than replacing C-terminal membrane protein sequences [31]. One such sandwich fusion with an insertion site at the N-terminal end of a MalF cytoplasmic domain did indeed show behavior expected of cytoplasmically situated alkaline phosphatase (i.e., low activity). A disadvantage of using alkaline phosphatase sandwich fusions is that they can be quite toxic to cells producing them (Boyd, personal
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communication). An alternative approach that also leaves C-terminal membrane protein sequences intact with derivatives less prone to cause toxicity involves the insertion of short sequences whose subcellular locations can be easily determined (see Section 4).
3.6. Analysis of mammalian membrane proteins in E. coli
The topologies of several mammalian membrane proteins expressed in E. coli have been analyzed using gene fusions. The first protein to be analyzed was the [3-subunit of sheep kidney Na,K-ATPase, a protein with a very simple topology containing a single spanning sequence oriented with its N-terminal end facing the cytoplasm [32]. The properties of a set of 13-1actamase gene fusions were consistent with this topology, implying normal membrane insertion in E. coli. Three topologically complex mammalian membrane proteins have also been analyzed using alkaline phosphatase gene fusions. In all three cases, there was auxiliary evidence that at least some of the expressed protein molecules were functional when expressed in E. coli implying that they had attained their normal topologies and folded structures. For all three proteins, the fusion studies showed results compatible with the established mammalian cell topologies at some sites of the proteins but not at others. For example, in the analysis of the human ~2-adrenergic receptor, the fusion analysis correctly placed five extramembranous segments situated in the C-terminal part of the membrane-associated domain, but showed results inconsistent with the normal topology for the remaining three extramembranous segments in the N-terminal part of the protein [33]. The inconsistent behavior of the N-terminal fusions is probably due to the fact that the N-terminal region of the protein (which lacks a cleavable signal sequence) normally faces away from the cytoplasm, an arrangement known to give rise to anomalous results (see above). An AP fusion analysis of the first half of the mouse multidrug resistance (MDR) protein (corresponding to six transmembrane sequences) was also compatible with the proposed topology for most of the structure [34]. Interestingly, the analysis worked well for the first translocated segment, which contains the protein' s only normally glycosylated site. Since the glycosylation does not occur in E. coli, the success of the fusion analysis indicates that the absence of glycosylation does not alter the topology in this region of MDR. Analysis of AP fusions to the C-terminal region of MDR led to a proposal for a major revision of the protein's topology [35]. In a third case, the human erythrocyte glucose transporter (GLUT1), an AP fusion analysis yielded results similar to those found for the MDR protein, with fusions at the N-terminal half of the protein behaving largely as predicted from topology models, but with inconsistencies in the C-terminal region (Traxler and Beckwith, unpublished). The reasons for the difficulties in the analysis of MDR and GLUT1 have not been explored. However, in neither case was the proportion of functional molecules in the expressed forms determined, and it is possible that the proteins are inserted into the membrane of E. coli in a mixture of topologies, and that the activities of the hybrid proteins reflect this heterogeneity.
Membrane protein topology
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4. Analysis of membrane protein topology using inserted sites Most of the complications that arise in the analysis of membrane protein topology using gene fusions are due to the loss of C-terminal sequences in the hybrids analyzed. Several alternative approaches that do not suffer from this limitation are based on the insertion of easily identified target sites into the polypeptide. Examples include sequences subject to proteolysis, antibody binding, vectorial labeling, or endogenous modification. In these methods, site-directed mutagenesis is used to insert the target sequence (often after the elimination of interfering endogenous sites) into a specific segment of the protein, followed by a determination of the inserted sequence's subcellular location (Fig. 3). The overall topology of a membrane protein can be determined by assaying inserts at a number of different positions.
4.1. Proteolysis There is a substantial literature describing the use of proteolysis at naturally-occurring susceptible sites in a membrane protein to define the subcellular locations of the sites. However, the utility of this assay is generally limited by the rarity of naturally occurring protease-sensitive sites. For example, lactose permease was analyzed using chymotrypsin, trypsin, thermolysin, and papain treatments [36,37]. In each case, only a single cytoplasmic segment, about halfway along the length of the polypeptide, was efficiently cleaved. Molecular genetic methods have made it possible to overcome the limited distribution of naturally-occurring cleavage sites by the introduction of new sites. For example, in the case of leader peptidase (a bitopic membrane protein with one
periplasmic sequence periplasm........ ~
~
('~
l,Ili
cytoplasm ..,,J V ~J L A
target site ~
~'~r
.III
modification ('~
II!
"'Y V
k'J L
B
/"~
P,~'I ~[
I[I
1
""J V
k"J ~k',-
_
C
Fig. 3. Analysisof membrane protein topology using introduced target sequences. (A) A model of a protein's membrane topology is formulated and a specific segment of the protein (such as the periplasmic segment depicted as a bold line) is assigned a testable subcellular location. (B) Site-directed mutagenesis is used to alter the segment to include a target site that can be modified in a compartment-specific fashion, (e.g., a protease-sensitive site). (C) The assay is used to test the location of the segment. If the target site is modified (e.g., cleaved by a protease), then the segment bearing the site must be exposed to the compartment that contains the modifying agent.
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cytoplasmic segment), a trypsin-sensitive site was introduced into the cytoplasmic segment using a combination of deletion and insertion mutations. Peptide sequencing was used to prove that cleavage occurred at the expected site. In such constructions, it is crucial to identify the sites where cleavage occurs because a mutation generating a new cleavage site may also uncover sites elsewhere in the protein that are normally resistant to proteolysis. An example of such behavior was observed in studies of derivatives of the outer membrane protein OmpA [38]. OmpA mutants were constructed with trypsin cleavage sites at each of the three hydrophilic segments exposed to the periplasm. These mutant proteins were resistant to extracellular trypsin activity but were cleaved when both sides of the outer membrane were exposed to protease. One mutant, however, cleaved both at the newly introduced site and at a second position, indicating that a cryptic site had been uncovered. The mutation might cause misfolding which makes the cryptic site accessible to trypsin, or trypsin cleavage at the introduced site might make the cryptic site accessible. In such an approach, it is also important to demonstrate that mutations which introduce cleavage sites do not alter the topology. This difficulty was observed in studies in which lactose permease was modified by the insertion of a sequence capable of both biotinylation by endogenous enzymes and by cleavage by Factor Xa protease [39]. The insertion was introduced into periplasmic segments, but assays of both biotinylation and Factor Xa cleavage demonstrated that the mutant segments were retained in the cytoplasm. In a different study, lactose permease was mutagenized to introduce a trypsin cleavage site in the sixth periplasmic segment (Lee and Manoil, unpublished observations). The cleavage site was introduced by mutagenizing the lactose permease gene using a degenerate mixture of oligonucleotides encoding a lysine residue (recognized by trypsin) with a variety of adjacent sequences. This family of lactose permease derivatives containing potential cleavage sites was then screened for an insertion that both retained transport activity and was efficiently cleaved by trypsin in spheroplasts. This approach provides a simple systematic method to identify protease-sensitive derivatives of membrane proteins whose topology and folding are not significantly altered from that of wild-type.
4.2. Antibody binding The subcellular location of a segment within a membrane protein can also be identified after introducing an epitope by analyzing antibody binding. This method was used to study the large extramembranous domains in the c~l subunit of the human acetylcholine receptor (AChR) [40]. A recurring problem in the design of target site assays is finding regions within a membrane protein that will tolerate an insert. The authors of the AChR study showed that certain regions of the protein are poorly conserved between species and also have naturally occurring epitopes that are recognized in both native or denatured AChR. They reasoned that these regions probably do not have a tightly constrained conformation in native AChR, and are thus likely to tolerate insertions without causing major alterations in the protein's structure.
Membrane protein topology
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The study, which employed two different monoclonal antibodies, found that small insertions (five or eight residue epitopes) were poorly recognized by antibody compared to longer insertions or insertions in which the epitope was inserted in a tandem repeat. The use of extender sequences appears to have improved accessibility to the target sites, recognition of the target sites, or both.
4.3. Vectorial labeling A number of studies of membrane protein topology have utilized sulfhydryl reagents to modify selectively cysteine residues introduced into a protein by site-directed mutagenesis. This approach was introduced by Falke et al. in an analysis of the Tar chemoreceptor [41 ], and has also recently been used to analyze the MotA protein [42]. Cysteine residues situated in periplasmic segments of the proteins were rapidly modified by membrane-impermeant maleimide derivatives, whereas cysteine residues in cytoplasmic segments were slowly modified. For both proteins, the effects of the mutations on membrane insertion of the altered proteins could be assessed by analyzing swarming behavior. An extension of this approach was utilized in the analysis of the E. coli glucose 6-phosphate transporter [43]. In this study, transporter mutants containing cysteine residues introduced at different sites were modified with p-chloromercuribenzosulfonate, a sulfhydryl agent that is similar to substrate glucose 6-phosphate in both size and charge. The analysis allowed the authors not only to demonstrate the location of particular hydrophilic loops, but also to probe the structure of the pore through which glucose 6-phosphate enters cells. Cysteine residues have also been introduced into bacteriorhodopsin and modified with spin label reagents whose subcellular locations could be assayed using electron paramagnetic resonance. It was possible to distinguish residues buried inside the folded protein from residues exposed on the protein surface (to either the membrane or the aqueous environment) [44]. An extension of this method can distinguish between the membrane and aqueous environments and also provides a metric for how deeply a given surface residue is buried within the membrane [45].
4.4. Endogenous modification In eukaryotic cells, the glycosylation activity found in the lumen of the endoplasmic reticulum and Golgi can be used to modify target sites in a compartment-specific manner. In principle, potential glycosylation sites introduced into translocated sites but not cytoplasmic sites of a membrane protein are modified. The twelve transmembrane sequence model of the cystic fibrosis transmembrane conductance regulator (CFTR) was tested using this method [46]. The two known glycosylation sites in the fourth translocated segment were eliminated, and novel glycosylation sites were individually inserted into each of the other five putative translocated domains. When expressed in cells, all of the mutant proteins with sites in putative translocated domains were efficiently modified, a result which supports the CFTR topology model. All of the mutant proteins showed iodide efflux activities comparable to the wild type, implying that the modifications did not dramatically alter the topology or folding of the proteins.
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4.5. G e n e r a l c o n s i d e r a t i o n s
The assays described in this section are all complicated by the need for the inserted target site to be accessible to the modifying or binding agent. A target site within a hydrophilic segment may be poorly detectable due to steric hindrance from the segment itself, from neighboring hydrophilic segments, or from the lipid bilayer. The addition of 'extender' sequences has been useful in both the proteolytic and epitope binding assays to increase accessibility of target sites. In addition, the placement of a variety of different sequences on both sides of a target site makes it possible to screen for conformations that are efficiently recognized. A second concern in the use of such assays is that the introduction of target sites may alter the topology of the membrane protein being analyzed. However, if the mutant protein containing the introduced site exhibits a specific activity (e.g., for transport) which is the same as that of wild-type, then the topology of the mutant derivative is likely to be essentially normal.
5. Conclusion A variety of molecular genetic approaches are available for the analysis of membrane protein topology in prokaryotes and eukaryotes. Methods based on the generation and analysis of hybrid proteins are simple and generally reliable, but are subject to several difficulties due to the loss of C-terminal membrane protein sequences in the hybrid proteins generated. These difficulties can be overcome through the use of methods that involve the insertion of easily identifiable target sequences into the membrane protein sequence without loss of C-terminal sequences. However, such methods are somewhat laborious and are subject to technical difficulties of their own due to the requirements that the inserted sequences be accessible to external agents and that they not significantly alter the membrane topologies of the proteins modified. Abbreviations
AchR, al subunit of the human acetylcholine receptor AP, alkaline phosphatase CFTR, cystic fibrosis transmembrane conductance regulator GLUT1, human erythrocyte glucose transporter MDR, mouse multidrug resistance protein
References 1. 2. 3. 4. 5.
Jennings,M. (1989) Annu. Rev. Biochem. 58, 999-1027. Traxler,B., Boyd, D. and Beckwith, J. (1993)J. Membr. Biol. 132, 1-11. Boyd,D. (1994) in: Membrane Protein Structure, ed S. White. pp. 144-163. Oxford UniversityPress, New York. von Heijne, G. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 167-192. von Heijne, G. (1986) EMBO J. 5, 3021-3027.
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Molecular Genetic Analysis of Membrane Protein Topology M. LEE and C. MANOIL Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195, USA
9 1996 Elsevier Science B. V. All rights reserved
Handbook of Biological Physics Volume 2, edited by W.N. Konings, H.R. Kaback and J.S. Lolkema
189
Contents
1.
Introduction
2.
Construction of topology models based on amino acid sequences
3.
Assay of membrane protean topology using gene fusions . . . . . . . . . . . . . . . . . . . . .
4.
5.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................
3.1.
Generation of gene fusions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Assay of hybrid protein activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Evaluation of models
3.4.
Anomalous behavior of gene fusions in topology studies
3.5.
Alkaline phosphatase sandwich fusions
3.6.
Analysis of mammalian membrane proteins in E. coli . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................
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Analysis of membrane protein topology using inserted sites
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Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Antibody binding
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Vectorial labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Endogenous modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.5.
General considerations
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations
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:200
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
A fundamental aspect of integral membrane protein structure is the transmembrane topology of the polypeptide chain, i.e., the disposition of the chain relative to the lipid bilayer in which it is embedded. A variety of biochemical techniques have been employed to elucidate topological structure, including proteolysis, side chain covalent modification, and epitope recognition [ 1]. However, these techniques often are unable to provide a full topological description of a membrane protein because of their reliance on the natural occurrence of residues that are susceptible to proteolytic cleavage, have reactive side chains or are accessible to antibody binding. These limitations have been largely circumvented by the development of molecular genetic methods which can be used in conjunction with the more traditional approaches. Two molecular genetic strategies for analyzing membrane protein topology have been developed. The first relies on the properties of gene fusion-encoded hybrid proteins in which enzymes whose activities reflect subcellular localization are attached at different sites in a membrane protein. The second strategy depends on the introduction of sequences (e.g., protease cleavage sites) whose subcellular disposition can be easily assayed. In this chapter, we will review both approaches, with special reference to work that has analyzed Escherichia coli lactose permease as a model. The reader is also referred to several excellent earlier reviews [1-3].
2. Construction of topology models based on amino acid sequences
The first step in analyzing the topology of a membrane protein is usually the formulation of a model or models based on the amino acid sequence of the protein. The construction of such models is generally based on two assumptions. The first assumption is that membrane-spanning sequences correspond to apolar cx-helical sequences that are sufficiently long to span the bilayer. The second assumption is that the spanning sequences are oriented in accordance with the 'positive inside' rule [4], that is, the favored orientation maximizes the exposure of positive residues in hydrophilic loops facing the cytoplasmic side of the membrane. The major difficulties encountered in formulating topology models using these rules are: (1) some spanning sequences contain polar residues which can make their identification problematic; (2) large extramembranous domains do not follow the positive inside rule [5], and (3) non-helical spanning sequences, such as 13-strands and sequences which insert into the membrane without spanning it, may contribute to a protein's membrane association [6-9]. Nevertheless, most of the integral 191
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membrane protein structures that have been determined are in reasonable accord with these general rules [7,10,11 ].
3. Assay of membrane protein topology using gene fusions The logic of using gene fusions to analyze membrane protein topology is illustrated in Fig. 1 [12]. Fusions of a gene encoding a protein whose enzymatic activity reflects its subcellular location (e.g., E. c o l i alkaline phosphatase (AP) lacking its signal sequence) are generated at multiple sites in the gene for a membrane protein, and the activities of the resulting hybrid proteins are compared to the previously formulated models. In principle, the activity of each hybrid protein reveals whether the junction site normally faces toward or away from the cytoplasm. A number of different proteins have been used as subcellular location 'sensors' in this type of analysis. In bacteria, the most commonly used sensor proteins are alkaline phosphatase and 13-1actamase (which require export to the periplasm for activity) and [3-galactosidase (which requires cytoplasmic localization for maximal activity) [2]. The combined use of periplasmic-active and cytoplasmic-active fusions provides positive activity signals for domains on both sides of the membrane, allowing a particularly thorough analysis (e.g., see Ref. [13]). A gene fusion analysis of topology generally consists of three steps: (1) the generation of gene fusions, (2) the assay of hybrid protein enzymatic activity, (3) a comparison of results to topology models.
1
IJ U1111 Ut
MEMBRANE PROTEIN
,,
ACTIVE ALKALINE PHOSPHATASE HYBRID
INACTIVE ALKALINE PHOSPHATASE HYBRID
Fig. 1. Analysis of membrane protein topology using alkaline phosphatase gene fusions. The top panel represents a membrane protein with six membrane-spanning segments. Gene fusions with fusion junctions corresponding to periplasmic positions (e.g., position 1) form hybrids with an exported alkaline phosphatase (AP) attached to N-terminal membrane protein sequences (bottom, left) which exhibit AP activity. Fusions at cytoplasmic positions (e.g., position 2) generate a cytoplasmically disposed AP which is inactive. Cytoplasmic AP appears to be inactive because it cannot form disulfide bonds necessary for folding [47].
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3.1. Generation of gene fusions Two methods have been commonly used for the generation of gene fusions: the insertion of transposon derivatives that 'automatically' generate the fusions, and in vitro mutagenesis using oligonucleotide-based methods. Both approaches employ membrane protein genes carried on plasmids. The two methods have different advantages and limitations. The generation of fusions by transposition is technically simple but suffers from the disadvantage that the junction sites are determined by the specificity of transposon insertion [ 14]. Although this difficulty is minimized for transposon derivatives with a relatively low insertion specificity (e.g., derivatives based on Tn5) [15], it may still be difficult to identify fusions with junctions in small extramembranous domains, which are common in transport proteins. The generation of fusions with predefined junctions using site-directed mutagenesis [16] or Polymerase Chain Reaction-based methods [17] is more laborious than generating the fusions by transposition, but has the advantage that junction positions can be specified by the oligonucleotides employed. This advantage is especially useful in distinguishing between closely related models for a structure (e.g., see Ref. [18]). Additional methods for generating fusions based on the interconversion of different fusion types ('fusion switching') [19] and the generation of nested deletions in appropriate plasmid constructs [20] have also been developed. The fusion switching technique makes it possible to compare the activities of hybrid proteins with different sensor proteins attached at exactly the same site in the membrane protein. The junction sites of fusions generated using any of these methods can be identified simply by DNA sequence analysis using oligonucleotide primers complementary to sequences encoding the 'sensor' proteins [21 ].
3.2. Assay of hybrid protein activity The enzyme activities of hybrid proteins are typically determined by assay of permeabilized cells using chromogenic substrates (alkaline phosphatase or I]-galactosidase) [21 ] or by means of a bioassay (I]-lactamase) [22]. Since gene fusions with different junction positions may be expressed at different levels, it is essential that rates of hybrid protein synthesis are examined to correct for such differences [23].
3.3. Evaluation of models The final step in a topology analysis is a comparison of results with models predicted from the sequence. As an example, the results of an AP gene fusion analysis of E. coli lactose permease are shown (Fig. 2). The fusions were generated by a combination of transposon TnphoA insertion and in vitro mutagenesis [ 18,24]. The activities of most fusions are consistent with the topology model, with periplasmic fusions expressing high alkaline phosphatase activities and cytoplasmic fusions expressing lower activities. Many of the lactose permease-alkaline phosphatase fusion junctions fall into the putative membrane-spanning sequences. A study of fusions with junctions within spanning sequences oriented with their N-termini facing the cytoplasm
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PERI
MEM
CYT
Fig. 2. Alkalinephosphatase gene fusion analysis of lactose permease. The positions and activities of AP gene fusions are shown [ 18,24] on the twelve-span model for the topologyof lactose permease. Highly active fusions are represented as filled arrowheads, partially active fusions as cross-hatched arrowheads, and inactive fusions as open arrowheads. PERI, periplasm; MEM, membrane; CYT, cytoplasm. (spanning sequences 3, 5, 7 and 11) indicates that fusions carrying more than 9-1 1 residues of the spanning sequence express maximal AP activities. It thus appears that only about half of a spanning sequence suffices for the protein export function of a membrane-spanning sequence. This conclusion is consistent with a variety of other genetic studies of protein export signals [25]. Fusions with junctions in spanning sequences in the opposite orientation (N end periplasmic) tend to show elevated alkaline phosphatase activities unless they contain not only the entire apolar sequence but C-terminal charged residues as well. This requirement evidently accounts for the partial AP activities of the fusions in cytoplasmic segment 3 (indicated by cross-hatched arrowheads). Detailed studies of analogous alkaline phosphatase fusions to MalF protein first implied a special role for positively charged residues in the cytoplasmic localization of such domains [26]. An amphipathic sequence plays an apparently analogous role in the E. c o l i serine chemoreceptor [27]. Two other classes of anomalous fusions have been defined and are discussed below (Section 3.4). The overall success of gene fusion methods in analyzing membrane protein topology implies that the subcellular localization of a site in a membrane protein does not, in general, require C-terminal sequences. The information dictating a
Membrane protein topology
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particular topology must therefore be present in sequences N-terminal to a given site or redundantly determined by N-terminal and C-terminal sequences. In addition, the folding defects caused by the loss of C-terminal membrane protein sequences in hybrid proteins evidently does not usually prevent the remaining N-terminal sequences from adopting the wild-type topology.
3.4. Anomalous behavior in topology studies Three types of anomalous behavior have been noted in topology studies involving alkaline phosphatase fusions. As mentioned in the previous section, alkaline phosphatase fusions with junctions near the N-terminal end of a cytoplasmic loop in a membrane protein can exhibit anomalously high activity. The unexpectedly high activity of such fusions may be due to loss of C-terminal hydrophilic sequences such as positive residues required to orient the preceding transmembrane sequence. A recent study of one such fusion site in MalF indicated that 13-1actamase fusions may be less prone to this type of anomaly than AP fusions [28]. A second anomaly occasionally occurs when periplasmic domain AP fusions exhibit unusually low activities (e.g., the fusion to the fifth periplasmic domain of lactose permease) (Fig. 2) [ 18,29]. These cases involve fusions with junction points C-terminal to spanning sequences carrying charged residues. The charged residues reduce the overall hydrophobicity of the spanning sequence and may decrease the sequence' s ability to promote export. In such cases, C-terminal spanning sequences in the unfused protein may be required for efficient export of the hydrophilic domain in question. Since such charged residues in spanning sequences are often important in the biological function of membrane proteins, anomalously low alkaline phosphatase activities may commonly be observed for fusions with junctions in regions of functional importance (e.g., regions in a transport protein involved in binding hydrophilic substrates during passage across the membrane). A third anomaly in gene fusion analysis of topology is encountered for membrane proteins whose (unprocessed) N-termini face the periplasm [13,30]. Hybrid proteins with junctions in these N-terminal domains lack transmembrane sequences and are, in general, not exported.
3.5. Alkaline phosphatase sandwich fusions The anomalies in gene fusion analysis of membrane protein topology are thought to arise at sites where C-terminal sequences are required for normal subcellular localization. In an attempt to circumvent this problem, a novel type of alkaline phosphatase fusion ('sandwich fusion') was developed in which the alkaline phosphatase sequence is inserted into the membrane protein sequence rather than replacing C-terminal membrane protein sequences [31]. One such sandwich fusion with an insertion site at the N-terminal end of a MalF cytoplasmic domain did indeed show behavior expected of cytoplasmically situated alkaline phosphatase (i.e., low activity). A disadvantage of using alkaline phosphatase sandwich fusions is that they can be quite toxic to cells producing them (Boyd, personal
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communication). An alternative approach that also leaves C-terminal membrane protein sequences intact with derivatives less prone to cause toxicity involves the insertion of short sequences whose subcellular locations can be easily determined (see Section 4).
3.6. Analysis of mammalian membrane proteins in E. coli
The topologies of several mammalian membrane proteins expressed in E. coli have been analyzed using gene fusions. The first protein to be analyzed was the [3-subunit of sheep kidney Na,K-ATPase, a protein with a very simple topology containing a single spanning sequence oriented with its N-terminal end facing the cytoplasm [32]. The properties of a set of 13-1actamase gene fusions were consistent with this topology, implying normal membrane insertion in E. coli. Three topologically complex mammalian membrane proteins have also been analyzed using alkaline phosphatase gene fusions. In all three cases, there was auxiliary evidence that at least some of the expressed protein molecules were functional when expressed in E. coli implying that they had attained their normal topologies and folded structures. For all three proteins, the fusion studies showed results compatible with the established mammalian cell topologies at some sites of the proteins but not at others. For example, in the analysis of the human ~2-adrenergic receptor, the fusion analysis correctly placed five extramembranous segments situated in the C-terminal part of the membrane-associated domain, but showed results inconsistent with the normal topology for the remaining three extramembranous segments in the N-terminal part of the protein [33]. The inconsistent behavior of the N-terminal fusions is probably due to the fact that the N-terminal region of the protein (which lacks a cleavable signal sequence) normally faces away from the cytoplasm, an arrangement known to give rise to anomalous results (see above). An AP fusion analysis of the first half of the mouse multidrug resistance (MDR) protein (corresponding to six transmembrane sequences) was also compatible with the proposed topology for most of the structure [34]. Interestingly, the analysis worked well for the first translocated segment, which contains the protein' s only normally glycosylated site. Since the glycosylation does not occur in E. coli, the success of the fusion analysis indicates that the absence of glycosylation does not alter the topology in this region of MDR. Analysis of AP fusions to the C-terminal region of MDR led to a proposal for a major revision of the protein's topology [35]. In a third case, the human erythrocyte glucose transporter (GLUT1), an AP fusion analysis yielded results similar to those found for the MDR protein, with fusions at the N-terminal half of the protein behaving largely as predicted from topology models, but with inconsistencies in the C-terminal region (Traxler and Beckwith, unpublished). The reasons for the difficulties in the analysis of MDR and GLUT1 have not been explored. However, in neither case was the proportion of functional molecules in the expressed forms determined, and it is possible that the proteins are inserted into the membrane of E. coli in a mixture of topologies, and that the activities of the hybrid proteins reflect this heterogeneity.
Membrane protein topology
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4. Analysis of membrane protein topology using inserted sites Most of the complications that arise in the analysis of membrane protein topology using gene fusions are due to the loss of C-terminal sequences in the hybrids analyzed. Several alternative approaches that do not suffer from this limitation are based on the insertion of easily identified target sites into the polypeptide. Examples include sequences subject to proteolysis, antibody binding, vectorial labeling, or endogenous modification. In these methods, site-directed mutagenesis is used to insert the target sequence (often after the elimination of interfering endogenous sites) into a specific segment of the protein, followed by a determination of the inserted sequence's subcellular location (Fig. 3). The overall topology of a membrane protein can be determined by assaying inserts at a number of different positions.
4.1. Proteolysis There is a substantial literature describing the use of proteolysis at naturally-occurring susceptible sites in a membrane protein to define the subcellular locations of the sites. However, the utility of this assay is generally limited by the rarity of naturally occurring protease-sensitive sites. For example, lactose permease was analyzed using chymotrypsin, trypsin, thermolysin, and papain treatments [36,37]. In each case, only a single cytoplasmic segment, about halfway along the length of the polypeptide, was efficiently cleaved. Molecular genetic methods have made it possible to overcome the limited distribution of naturally-occurring cleavage sites by the introduction of new sites. For example, in the case of leader peptidase (a bitopic membrane protein with one
periplasmic sequence periplasm........ ~
~
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1
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Fig. 3. Analysisof membrane protein topology using introduced target sequences. (A) A model of a protein's membrane topology is formulated and a specific segment of the protein (such as the periplasmic segment depicted as a bold line) is assigned a testable subcellular location. (B) Site-directed mutagenesis is used to alter the segment to include a target site that can be modified in a compartment-specific fashion, (e.g., a protease-sensitive site). (C) The assay is used to test the location of the segment. If the target site is modified (e.g., cleaved by a protease), then the segment bearing the site must be exposed to the compartment that contains the modifying agent.
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cytoplasmic segment), a trypsin-sensitive site was introduced into the cytoplasmic segment using a combination of deletion and insertion mutations. Peptide sequencing was used to prove that cleavage occurred at the expected site. In such constructions, it is crucial to identify the sites where cleavage occurs because a mutation generating a new cleavage site may also uncover sites elsewhere in the protein that are normally resistant to proteolysis. An example of such behavior was observed in studies of derivatives of the outer membrane protein OmpA [38]. OmpA mutants were constructed with trypsin cleavage sites at each of the three hydrophilic segments exposed to the periplasm. These mutant proteins were resistant to extracellular trypsin activity but were cleaved when both sides of the outer membrane were exposed to protease. One mutant, however, cleaved both at the newly introduced site and at a second position, indicating that a cryptic site had been uncovered. The mutation might cause misfolding which makes the cryptic site accessible to trypsin, or trypsin cleavage at the introduced site might make the cryptic site accessible. In such an approach, it is also important to demonstrate that mutations which introduce cleavage sites do not alter the topology. This difficulty was observed in studies in which lactose permease was modified by the insertion of a sequence capable of both biotinylation by endogenous enzymes and by cleavage by Factor Xa protease [39]. The insertion was introduced into periplasmic segments, but assays of both biotinylation and Factor Xa cleavage demonstrated that the mutant segments were retained in the cytoplasm. In a different study, lactose permease was mutagenized to introduce a trypsin cleavage site in the sixth periplasmic segment (Lee and Manoil, unpublished observations). The cleavage site was introduced by mutagenizing the lactose permease gene using a degenerate mixture of oligonucleotides encoding a lysine residue (recognized by trypsin) with a variety of adjacent sequences. This family of lactose permease derivatives containing potential cleavage sites was then screened for an insertion that both retained transport activity and was efficiently cleaved by trypsin in spheroplasts. This approach provides a simple systematic method to identify protease-sensitive derivatives of membrane proteins whose topology and folding are not significantly altered from that of wild-type.
4.2. Antibody binding The subcellular location of a segment within a membrane protein can also be identified after introducing an epitope by analyzing antibody binding. This method was used to study the large extramembranous domains in the c~l subunit of the human acetylcholine receptor (AChR) [40]. A recurring problem in the design of target site assays is finding regions within a membrane protein that will tolerate an insert. The authors of the AChR study showed that certain regions of the protein are poorly conserved between species and also have naturally occurring epitopes that are recognized in both native or denatured AChR. They reasoned that these regions probably do not have a tightly constrained conformation in native AChR, and are thus likely to tolerate insertions without causing major alterations in the protein's structure.
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The study, which employed two different monoclonal antibodies, found that small insertions (five or eight residue epitopes) were poorly recognized by antibody compared to longer insertions or insertions in which the epitope was inserted in a tandem repeat. The use of extender sequences appears to have improved accessibility to the target sites, recognition of the target sites, or both.
4.3. Vectorial labeling A number of studies of membrane protein topology have utilized sulfhydryl reagents to modify selectively cysteine residues introduced into a protein by site-directed mutagenesis. This approach was introduced by Falke et al. in an analysis of the Tar chemoreceptor [41 ], and has also recently been used to analyze the MotA protein [42]. Cysteine residues situated in periplasmic segments of the proteins were rapidly modified by membrane-impermeant maleimide derivatives, whereas cysteine residues in cytoplasmic segments were slowly modified. For both proteins, the effects of the mutations on membrane insertion of the altered proteins could be assessed by analyzing swarming behavior. An extension of this approach was utilized in the analysis of the E. coli glucose 6-phosphate transporter [43]. In this study, transporter mutants containing cysteine residues introduced at different sites were modified with p-chloromercuribenzosulfonate, a sulfhydryl agent that is similar to substrate glucose 6-phosphate in both size and charge. The analysis allowed the authors not only to demonstrate the location of particular hydrophilic loops, but also to probe the structure of the pore through which glucose 6-phosphate enters cells. Cysteine residues have also been introduced into bacteriorhodopsin and modified with spin label reagents whose subcellular locations could be assayed using electron paramagnetic resonance. It was possible to distinguish residues buried inside the folded protein from residues exposed on the protein surface (to either the membrane or the aqueous environment) [44]. An extension of this method can distinguish between the membrane and aqueous environments and also provides a metric for how deeply a given surface residue is buried within the membrane [45].
4.4. Endogenous modification In eukaryotic cells, the glycosylation activity found in the lumen of the endoplasmic reticulum and Golgi can be used to modify target sites in a compartment-specific manner. In principle, potential glycosylation sites introduced into translocated sites but not cytoplasmic sites of a membrane protein are modified. The twelve transmembrane sequence model of the cystic fibrosis transmembrane conductance regulator (CFTR) was tested using this method [46]. The two known glycosylation sites in the fourth translocated segment were eliminated, and novel glycosylation sites were individually inserted into each of the other five putative translocated domains. When expressed in cells, all of the mutant proteins with sites in putative translocated domains were efficiently modified, a result which supports the CFTR topology model. All of the mutant proteins showed iodide efflux activities comparable to the wild type, implying that the modifications did not dramatically alter the topology or folding of the proteins.
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4.5. G e n e r a l c o n s i d e r a t i o n s
The assays described in this section are all complicated by the need for the inserted target site to be accessible to the modifying or binding agent. A target site within a hydrophilic segment may be poorly detectable due to steric hindrance from the segment itself, from neighboring hydrophilic segments, or from the lipid bilayer. The addition of 'extender' sequences has been useful in both the proteolytic and epitope binding assays to increase accessibility of target sites. In addition, the placement of a variety of different sequences on both sides of a target site makes it possible to screen for conformations that are efficiently recognized. A second concern in the use of such assays is that the introduction of target sites may alter the topology of the membrane protein being analyzed. However, if the mutant protein containing the introduced site exhibits a specific activity (e.g., for transport) which is the same as that of wild-type, then the topology of the mutant derivative is likely to be essentially normal.
5. Conclusion A variety of molecular genetic approaches are available for the analysis of membrane protein topology in prokaryotes and eukaryotes. Methods based on the generation and analysis of hybrid proteins are simple and generally reliable, but are subject to several difficulties due to the loss of C-terminal membrane protein sequences in the hybrid proteins generated. These difficulties can be overcome through the use of methods that involve the insertion of easily identifiable target sequences into the membrane protein sequence without loss of C-terminal sequences. However, such methods are somewhat laborious and are subject to technical difficulties of their own due to the requirements that the inserted sequences be accessible to external agents and that they not significantly alter the membrane topologies of the proteins modified. Abbreviations
AchR, al subunit of the human acetylcholine receptor AP, alkaline phosphatase CFTR, cystic fibrosis transmembrane conductance regulator GLUT1, human erythrocyte glucose transporter MDR, mouse multidrug resistance protein
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