©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Artificial Transmembrane Segments
REQUIREMENTS FOR STOP TRANSFER AND POLYPEPTIDE ORIENTATION (*)

Huanfeng Chen , Debra A. Kendall (§)

From the (1) Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transmembrane segments of proteins generally consist of a long stretch of hydrophobic amino acids, which can function to initiate membrane insertion (start-stop sequences), initiate translocation (signal-anchor sequences), or stop further translocation of the following polypeptide chain (stop-transfer sequences). In this study, we have taken Escherichia coli alkaline phosphatase, a transported and water-soluble protein, and examined the requirements for converting it into a transmembrane protein with particular orientation. Since the wild type enzyme is transported, there is no predisposition against membrane translocation, yet it is not a membrane protein, so it does not possess any intrinsic membrane topogenic preferences. A series of potential transmembrane segments was introduced into an internal position of the enzyme to test the ability of each to initiate translocation, stop translocation, and adopt a particular orientation. For this purpose, cassette mutagenesis was used to incorporate new structural segments composed of polymers of alanines and leucines. The threshold value of hydrophobicity required to function as a stop-transfer sequence was determined. For a transmembrane segment of typical length (21 residues), this value is equivalent to the hydrophobicity of 16 alanines and 5 leucines. Interestingly, much shorter segments will also suffice to stop translocation, but these must be composed of more highly hydrophobic residues (e.g. 11 leucines). When the wild type amino-terminal signal peptide is deleted or made dysfunctional, sufficiently hydrophobic internal segments can initiate translocation of the following polypeptide and function as a signal anchor. Furthermore, in so doing, the orientation of the protein is changed from N-C to N-C


INTRODUCTION

For integral membrane proteins, the problem of obtaining structural data has proven to be very complex, and only a handful of structures have been solved to high resolution (Roth et al., 1989; Henderson et al., 1990; Schertler et al., 1993). At the same time, the identification of new membrane proteins and their corresponding primary sequences is moving at a rapid pace. Consequently, construction of topological models often relies on sequence analysis to identify hydrophobic stretches of about 20 residues as putative transmembrane domains and clusters of positively charged residues to predict orientation. While this approach has proven remarkably consistent with experimentally defined topological data (von Heijne and Gavel, 1988; von Heijne, 1992), nonclassical transmembrane domains may be overlooked because little is known regarding the limits of variation in membrane protein structure.

Theoretically, hydrophobic -helical segments of at least 20 residues are required to provide a hydrophobic surface of sufficient length to traverse the normal 30-Å thickness of the hydrophobic interior of a bilayer (Tanford, 1978). Indeed, the natural transmembrane segments of glycophorin A (Tomita et al., 1978) and of the coliphage f1 gene II protein (Davis et al., 1985) are 23 residues in length, and that of the vesicular stomatitis virus glycoprotein (G) contains 20 residues (Adams and Rose, 1985). However, truncated natural transmembrane segments and their replacement with artificial sequences with as few as 12 nonpolar residues can be effective stop-transfer sequences (Davis et al., 1985; Davis and Model, 1985; Spiess and Handschin, 1987). Conversely, some long hydrophobic segments fail to stop translocation, such as the fusion-related hydrophobic domains from eukaryotic viruses, which can be passed through the host cell membranes and the cytoplasmic membrane of Escherichia coli (Hsu et al., 1983; Davis and Hsu, 1986).

Positive charges in the region flanking a transmembrane segment have been shown to have significant effects on the orientation of a transmembrane segment. The ``positive inside'' hypothesis (von Heijne, 1986, 1992) suggests that positive charges (or the charge differential, Hartmann et al., 1989) surrounding the transmembrane segments are the predominant topogenic signals that determine the orientations of the transmembrane segments (Popot and Engelman, 1990; Andersson and von Heijne, 1994). The positive charges are assumed to present an energy barrier to translocation and, therefore, hold the transmembrane segment in such an orientation that the positive charges remain inside the cytoplasm (Andersson and von Heijne, 1994). The orientation of many natural transmembrane segments is consistent with this hypothesis (von Heijne and Gavel, 1988), yet counterexamples have also been observed (Audigier et al., 1987; Hartmann et al., 1989; Parks and Lamb, 1991; Andrews et al., 1992; Locker et al., 1992). In addition to the effect of charges in flanking regions, there is also evidence that the amino-terminal (Andrews et al., 1992), carboxyl-terminal (Bibi et al., 1991), or the transmembrane regions themselves (Parks et al., 1989; Locker et al., 1992) may influence or direct the orientation of a transmembrane segment.

In this study, we have taken E. coli alkaline phosphatase, a normally transported and water-soluble protein, and examined the requirements for converting it into a transmembrane protein with particular orientation. This approach has several advantages: the globular form of the enzyme is active in the periplasm but not the cytoplasm of E. coli (Michaelis et al., 1986), which provides a useful indicator of the extent to which mutants become anchored to the membrane. The structure of the enzyme has been determined to high resolution (Kim and Wyckoff, 1989, 1991), and this provides the basis for choosing locations to insert potential transmembrane segments. In E. coli, the wild type form is periplasmic, so there should be no predisposition against membrane translocation, and since it is not a membrane protein, it does not possess any intrinsic membrane topogenic preferences. By inserting polymeric sequences of different length and hydrophobicity into an internal position of the mature protein, we have defined a threshold value of hydrophobicity required to stop protein translocation and function as a membrane anchor. Surprisingly, very short highly hydrophobic segments can provide stable membrane anchors. Furthermore, an internal hydrophobic segment of appropriate hydrophobicity can adopt different orientations, depending on the export competence of the amino-terminal signal peptide.


MATERIALS AND METHODS

Bacterial Strains and Media

E. coli AW1043 (lac galU galK (leu-ara)phoA-E15 proC::Tn5) was used for the generation and replication of mutant forms of alkaline phosphatase and for all transport and membrane association analyses. For mutagenesis and general propagation of bacterial strains, LB medium (Sambrook et al., 1989) supplemented with 50 µg/ml kanamycin and 250 µg/ml ampicillin was used. For transport and membrane association analyses, cells were cultured in MOPS() medium containing 50 µg/ml kanamycin and 250 µg/ml ampicillin.

DNA Manipulations and Mutagenesis

The CN-A, CN-AA, CN-L, CN-15L, and CN-LL mutants were generated using the plasmid, CN. CN is a modified form of WT CASS3, which is a pBR322 derivative containing the alkaline phosphatase gene (phoA) and its natural promotor (Kendall and Kaiser, 1988). A unique NcoI site is located at codons 275-277 of the region encoding mature alkaline phosphatase. An additional unique site, HpaI, was introduced at the original NcoI site by ligating a small double-stranded oligonucleotide fragment carrying the HpaI site to NcoI-digested CASS3. The resulting plasmid, CN, has four additional codons (encoding Leu, Thr, His, and Gly) at that position and contains an additional NcoI site. Mutant sequences were generated by ligation of oligonucleotides with HpaI-digested CN vector as previously described (Chou and Kendall, 1990). For the leucine series, single or multiple copies of oligonucleotides coding for five leucines were inserted at the HpaI site. For the alanine series, an oligonucleotide insert coding for five alanines and carrying a unique SacII site was first introduced in the HpaI site. This removed the HpaI site, but longer alanine sequences were generated by inserting additional copies of five alanine segments into the SacII site. The design and sequences of the oligonucleotides used here are described in Chou and Kendall(1990) and Doud et al.(1993).

For the CN-LA1, CN-LA2, and CN-LA3 mutants, a linker containing the unique sites XhoI and HpaI was inserted into the NcoI site of the WT CASS3 plasmid. The resulting plasmid, CN-B, was then digested with XhoI and HpaI to remove the intervening linker sequence, and the large vector was purified. Oligonucleotide inserts coding for various combinations of alanine and leucine residues were then ligated to this vector. The mutants CN-LA1, CN-LA2, and CN-LA3 have an additional serine and threonine residue at the beginning of the inserted hydrophobic segment relative to the CN vector-derived mutants. XhoI and SalI produce cohesive protruding ends with the same sequence, and, therefore, oligonucleotides designed to ligate to SalI-treated vectors can also be ligated to this XhoI-treated vector.

The DS-CN and DS-LL mutants were generated by deleting 16 amino acids in the amino-terminal signal peptide, including the entire hydrophobic core and cleavage regions. CN and CN-LL were digested with SalI and BssHII. The recessed 3` termini that were generated were then filled in with Klenow, and the blunt ends were ligated. The resulting mutants have the initiating methionine, lysine, glutamine, serine, and threonine followed by the sequence of the mature region of alkaline phosphatase. The 7A-LL, 6A-LL, and 2A-LL mutants were constructed by replacing the amino-terminal signal peptide hydrophobic core region of CN-LL with various ratios of alanine to leucine residues (7A3L, 6A4L, and 2A8L). These signal peptide mutant sequences were previously generated (Doud et al., 1993) and only needed to be exchanged with the complementary wild type signal peptide region of the CN-LL vector.

Two molecular weight markers, TLF and TSF, were also constructed for this study. The TLF marker was generated by digesting the WT CASS3 plasmid with NcoI, filling the recessed 3` termini with Klenow and then ligating the blunt ends. This resulted in a frameshift and introduced five new amino acids after amino acid 278 followed by a stop codon. To generate TSF, equal amounts of the CN vector and mutant 10L (Chou and Kendall, 1990) were digested together with HpaI and BamHI, extracted, and then ligated together. A product was isolated in which the amino acids from -6 to 278 of alkaline phosphatase were deleted. The truncated alkaline phosphatase (TSF) contains most of the signal peptide and the remaining carboxyl-terminal tail of the enzyme (original residues from 278 to 449).

Induction of Alkaline Phosphatase Expression

Overnight cultures grown in LB medium containing 250 µg/ml ampicillin and 50 µg/ml kanamycin were subcultured 1:20 in the same medium. Cells were then grown at 37 °C with vigorous shaking to logarithmic phase and harvested at A 1.0-1.2. An aliquot of cells was then taken and washed twice with MOPS containing no phosphate and resuspended in 2 ml of the same medium supplemented with amino acids (20 µg/ml, minus methionine). Cells were incubated at 37 °C for 15 min with shaking to induce the expression of alkaline phosphatase.

Alkaline Phosphatase Activity Assays

Alkaline phosphatase is enzymatically active only when it is transported to the periplasm or membrane bound with the bulk of the enzyme in a periplasmic orientation (Michaelis et al., 1986; Manoil and Beckwith, 1985). After induction of alkaline phosphatase, cells were lysed and treated with the substrate p-nitrophenyl phosphate as described by Brickman and Beckwith(1975) and Michaelis et al.(1986). The absorbance at 410 nm was used to determine the extent of hydrolysis of p-nitrophenyl phosphate to p-nitrophenol by alkaline phosphatase.

Cell Fractionation

Following the induction of alkaline phosphatase, cells were radiolabeled with 40 µCi/ml L-[S]methionine for 30 s at 37 °C, then chased with cold methionine at a final concentration of 4 mg/ml for 30 s, and then quickly cooled on ice. One-half of the cells were precipitated with trichloroacetic acid to give a ``whole cell'' sample. The remaining cells were pelleted. The supernatant was discarded, and the pellet was resuspended in 0.1 M Tris, pH 8.2, 0.5 M sucrose, 0.5 mM EDTA and treated with freshly prepared lysozyme. Immediately following, an equal amount of ice-cold water was added and mixed well, and the mixture was incubated for 5 min on ice. To stabilize the forming spheroplasts, Mg was added to a final concentration of 10 mM during the incubation (Thom and Randall, 1988). The cells were then centrifuged to separate the periplasmic contents from the spheroplasts. The periplasmic fraction was precipitated with trichloroacetic acid and subjected to immunoprecipitation of alkaline phosphatase as described by Kendall et al.(1986).

NaOH Treatment

Following labeling as described above, an aliquot of cells was removed to an ice-cold tube, and an equal volume of ice-cold 0.1 M NaOH solution was added (Russel and Model, 1982). The mixture was vortexed hard and then centrifuged at 4 °C for 10 min in a microcentrifuge to separate the soluble fraction (supernatant) and membrane-associated proteins (pellet). The protein in each fraction was then precipitated with trichloroacetic acid, and alkaline phosphatase was immunoprecipitated as described.

Flotation Gradient Centrifugation

Flotation experiments were performed as described by Thom and Randall(1988) on [S]methionine-labeled cells. After centrifugation, the contents of the tube were separated into six 150-µl fractions, each taken successively from the top of the gradient. The protein was then precipitated with trichloroacetic acid and analyzed by autoradiography.

Protease Accessibility Analysis

After labeling and converting cells to spheroplasts as described above, cells were divided into three fractions. One fraction of cells was precipitated with trichloroacetic acid to provide an untreated control. Proteinase K (final concentration, 25 µg/ml) was added to the other two fractions. CHAPS (final concentration, 0.2%) was added to one of the protease-treated fractions. Both fractions were incubated on ice for 15 min. Protein was precipitated with trichloroacetic acid after the incubation. Alkaline phosphatase and its partially digested fragments were then immunoprecipitated as described by Kendall et al. (1986).

Electrophoresis and Quantitation of Protein Bands

Immunoprecipitated proteins were separated by electrophoresis on 7.5-10% Laemmli SDS-polyacrylamide gel electrophoresis gels (Laemmli, 1970). The pattern was visualized by autoradiography as described by Kendall and Kaiser(1988), and protein was quantified by scintillation counting.


RESULTS

To examine the hydrophobicity and length requirements for a segment to halt polypeptide translocation and function as a stop transfer sequence, segments composed of different lengths of polyalanine, polyleucine, or mixtures of alanine and leucine residues were inserted into the mature region of alkaline phosphatase. Alanine and leucine were chosen for this study because both have a high -helical propensity (Ferretti and Paolillo, 1969; Arfmann et al., 1977; Chou and Fasman, 1978), and both have alkyl side chains, which are nonpolar but to very different degrees. Amino acid position 278 was chosen as the location for the insertions because it is in a loosely folded loop region, which is removed in the tertiary structure from the core of the enzyme and the catalytic site (Kim and Wyckoff, 1989, 1991). We anticipated that this region may be able to accommodate the introduction of relatively long segments with minimal disruption to the integrity of the globular structure and the activity of transported alkaline phosphatase. On the other hand, the generation of a transmembrane form should inhibit final folding and enzymatic activity. Fig. 1shows the amino acid sequences of the resulting mutants. Note that the mutants all contain an additional leucine immediately carboxyl to the insert and therefore contain a total of 11 or 21 contiguous hydrophobic residues. The ten-residue segments flanking the inserts have net charges of +2.5 (amino-terminal side) and +0.5 (carboxyl-terminal side), counting histidine as +0.5.


Figure 1: Amino acid sequences of the relevant regions of mutant alkaline phosphatases. Wild type CASS3 (Kendall and Kaiser, 1988) and CN, a derivative with one additional leucine residue, are also included for comparison. A, sequences of the mutants that have segments of polyalanine, polyleucine, or mixtures of leucines and alanines inserted at the HpaI site of the CN vector or the XhoI and HpaI sites of the CN-B vector. All mutants retained wild type signal peptides. The inserted hydrophobic residues are shown in boldfacetype. B, sequences of the mutants that have both internally inserted hydrophobic segments and a mutated signal peptide. Hydrophobic core regions of the signal peptide and the inserted hydrophobic segments are shown in boldfacetype. For mutants with deleted signal peptides, the deleted region is indicated by brackets. In all cases, an arrow marks the signal peptide cleavage site, and the numbersabove the sequences indicate the position of the amino acid in the entire mature wild type alkaline phosphatase sequence.



The transport properties of the mutants with polyalanine and polyleucine segments were first assessed by a cell fractionation experiment. As shown in Fig. 2, insertion of 10 or even 20 alanine residues did not stop protein translocation; the mutated proteins were mostly translocated and, like wild type alkaline phosphatase, released to the periplasm. In contrast, mutants containing insertions of either 10 or 20 leucine residues were not released to the periplasm, although their signal peptides were cleaved. Alkaline phosphatase activity levels are consistent with these findings. Wild type alkaline phosphatase is not active in the E. coli cytoplasm, but it becomes active subsequent to translocation, disulfide bond formation, and folding (Boyd et al., 1987; Derman and Beckwith, 1991). Mutants with the polyalanine inserts were found to be active, while no enzymatic activity was detected for the leucine series (Fig. 2).


Figure 2: Cell fractionation of mutants with various polyalanine or polyleucine segments. Cell fractionation studies were performed as described under ``Materials and Methods.'' Whole cell (W) and periplasmic fractions (P) are shown for each mutant. All bands correspond to the mature form of the protein. This was verified by the observation of precursor in the presence of azide. The aberrant mobility of CN-L likely reflects atypical SDS binding as has been seen before (Kendall et al., 1986; Vlasuk et al., 1984). The intensity of each band was quantified by liquid scintillation counting, and the percentage of alkaline phosphatase found in the periplasmic fraction is listed under each mutant. Also given are the enzymatic activities of each mutant. These were measured as described under ``Materials and Methods'' and normalized to CN, which was assigned a value of 100%.



Membrane localization of mutants containing the leucine segments was indicated by both NaOH extraction and flotation gradient centrifugation. The method of Russel and Model(1982) utilizes NaOH treatment of cells to separate membrane proteins (pellet) from cytoplasmic and periplasmic proteins (supernatant). This analysis (Fig. 3A) indicated that mutants with additional 10 or 20 leucines partitioned with the membrane fraction (84 and 94%, respectively), while those containing polyalanine segments remained associated with the soluble fraction (each only 2% in the membrane fraction). Gradient centrifugation of lysates from cells expressing the polyleucine mutants further revealed that these proteins float to the top of the gradient with the membranes, while a control mutant with neither a signal peptide nor a membrane-spanning segment (mutant DS-CN) largely remained at the bottom of the gradient with cytoplasmic proteins (Fig. 3B).


Figure 3: Membrane association of mutant proteins with internal polyleucine segments. A, NaOH extraction of mutant proteins. Cells expressing mutant proteins were treated with ice-cold NaOH immediately following the labeling. Pellets (M), which contain proteins stably associated with the membrane, were separated by centrifugation from the supernatant (S), which contains cytoplasmic, periplasmic, or loosely membrane-associated proteins. The intensity of the alkaline phosphatase bands in each fraction was quantitated by liquid scintillation counting, and the percentage of the membrane fraction was calculated with respect to the total counts of both fractions. B, flotation analysis of the mutant proteins. Spheroplasts of bacteria expressing the mutant proteins were generated and separated from the periplasmic fractions. Lysed spheroplasts were then subjected to centrifugation as described under ``Materials and Methods.'' After centrifugation, fractions 1-6 were removed sequentially from the top to the bottom of the centrifuge tube. Fractions 1 and 2 correspond to a 1.15-1.22 g/ml density of metrizamide and are considered membrane fractions. Fractions 5 and 6 correspond to a 1.3-1.4 g/ml density of metrizamide and are considered cytosolic fractions. Whole cell (W) and periplasmic contents (P) were also loaded for comparison.



A protease accessibility experiment (Fig. 4) revealed that the mutants with polyleucine inserts were membrane bound in an N-C orientation. Untranslocated alkaline phosphatase is sensitive to proteinase K because it is in a partially unfolded state; in contrast, the transported protein is resistant to protease because it is tightly folded into a more compact form. The wild type complement used in this study, mutant CN, has no hydrophobic segment insertion and, as expected, was insensitive to protease digestion even in the presence of detergent, which permeabilizes the cytoplasmic membrane. Mutants containing polyleucine inserts were however, completely digested in the presence of protease plus detergent, consistent with our earlier finding that these were not periplasmically localized. In contrast, when the protease was added in the absence of detergent, a partially protected fragment was evident, and the relative electrophoretic mobility of the fragment increased as the length of the inserted polyleucine segment increased. The results suggest that the protein is membrane-bound via the polyleucine segment in an N-C orientation. A comparison of the protected fragment and molecular weight markers revealed that the protected fragment corresponds to about one-third of the protein plus the length of the insertion (i.e. the carboxyl-terminal segment of the protein), and this is consistent with the N-C topological orientation depicted in Fig. 4 (bottom). Since mutants with polyalanine insertions were found to fractionate with the periplasm and retain enzymatic activity, they should be resistant to protease digestion. Indeed, the wild type domains of these mutants were resistant, but the inserted polyalanine portion was largely proteolyzed; little fully intact alkaline phosphatase was observed, but two fragments corresponding to molecular weight markers representing two-thirds and one-third of the protein (the lengths of the domains flanking the insert) were protease resistant even in the presence of detergent.


Figure 4: Orientation of membrane-associated proteins. Protease accessibility experiments were performed as described under ``Materials and Methods.'' Cytoplasmic or cytoplasmically oriented proteins are protected from digestion by added proteinase K in the absence of CHAPS but are exposed to the enzyme in the presence of the detergent. Periplasmic or periplasmically oriented proteins are exposed to proteinase K regardless of the presence of CHAPS. Wild-type alkaline phosphatase is transported to the periplasm and is tightly folded and resistant to proteinase K digestion. For the purpose of discussion, CN can be regarded as wild type and is resistant to proteinase K. CN-A and CN-AA are transported. However, the inserted alanine segments are in an exposed region and are digested by proteinase K (Chou and Kendall, 1990). The flanking domains of CN-A and CN-AA retain their compactness and are resistant to degradation. These are resolved essentially as two fragments that correspond with the molecular weight markers. Mutants CN-L, CN-15L, and CN-LL are anchored to the membrane in a N-C orientation as shown in the topology model at the bottom of the figure. The periplasmically oriented amino termini (Peri) are not tightly folded and are sensitive to proteinase K. The cytoplasmically oriented C termini (Cyto) are protected from proteinase K in the absence of CHAPS and digested when CHAPS is added. The molecular weight of the protected small C termini increases as the length of the inserted leucine segment increases. Molecular weight markers (TLF and TSF) were loaded in the firstlane, and their relative size with respect to wild type alkaline phosphatase is schematically shown by the filledbars next to their respective bands. Emptyarrowhead, TLF and the proteinase K-generated large amino-terminal fragments. Filledarrowhead, TSF and the proteinase K-generated small carboxyl fragments. All of the mutants exhibit signal peptide cleavage and are present in the mature form (m).



It is evident from a comparison of the properties of the mutants with polyalanine and polyleucine insertions that while there is considerable flexibility with regard to length, a segment must be sufficiently hydrophobic to function as a stop transfer segment. To determine the minimum level of hydrophobicity required for a stop transfer segment, a series of mutants was constructed, which varied in the ratio of alanine to leucine residues yet which retained an overall insertion length of 21 residues (see Fig. 1). The results of a cell fractionation experiment with these mutants are shown in Fig. 5 . Mutant CN-LA1 (3 leucines:18 alanines), which has the least hydrophobic insert, was translocated and released to the periplasm. Mutant CN-LA2 (5 leucines:16 alanines) represents a transition point with a marked reduction in transported alkaline phosphatase, and essentially no transported protein is detected for mutant CN-LA3 (7 leucines:14 alanines). The trend from transported to membrane-anchored forms was revealed further by NaOH extraction (Fig. 6). Stable membrane association distinctly increased with the higher leucine content. A protease accessibility experiment was consistent with these results and with the membrane-anchored form in the N-C orientation (data not shown), comparable with the mutants with the inserts of leucines only.


Figure 5: Cell fractionation of mutants with internal segments composed of alanine and leucine mixtures. Methods and identifications are as described in the legend to Fig. 2.




Figure 6: NaOH extraction of mutants with internal segments composed of alanine and leucine mixtures. Methods and identifications are as described in the legend to Fig. 3A.



The orientation of the membrane-bound form can be manipulated by changing the nature of upstream sequences. In particular, the effect of the amino-terminal signal sequence on the topology of an alkaline phosphatase mutant containing a polyleucine transmembrane segment was examined by signal peptide attenuation or deletion. Previous studies in our laboratory demonstrated that the function of the alkaline phosphatase signal peptide can be titrated by replacing the wild type hydrophobic core with different combinations of alanines and leucines (Doud et al., 1993). When the hydrophobicity of the signal peptide falls below the equivalent of 6:4 (alanines to leucines), transport is slow if it occurs at all, while at levels above the equivalent of 5:5 (alanines to leucines), the signal peptide is functional and transport is efficient. Mutants DS-LL, 7A-LL, 6A-LL, and 2A-LL were generated by deleting the signal peptide core region or replacing it with different ratios of alanine to leucine (see Fig. 1 ). In each case, the mature region of the protein corresponded to mutant CN-LL. The topological orientation of this series was analyzed by the protease accessibility method (Fig. 7). Interestingly, the molecular weight of the protected fragment shifted from one corresponding to the carboxyl-terminal domain (mutants 2A-LL and 6A-LL) to one corresponding to the amino-terminal domain (mutants 7A-LL and DS-LL). This suggests that the orientation changed from N-C to C-N when the signal peptide was changed from a functional to a dysfunctional one. In the presence of a functional signal peptide, the internal hydrophobic segment functions only to stop transfer of the translocating polypeptide chain (i.e. stop-transfer sequence). However, in the absence of a functional signal peptide, the precursor form is maintained, and the internal hydrophobic segment is itself capable of initiating membrane insertion; it does so in the same orientation that functional signal peptides do, and like a signal peptide, the portion of the protein carboxyl-terminal to it is translocated (i.e. it functions as a signal anchor).


Figure 7: Orientation of membrane-bound mutants with functional and dysfunctional signal peptides. The 6A-LL and 2A-LL mutants are anchored to the membrane in the N-C orientation as shown in the topology model at the bottom of the figure. The periplasmically oriented amino termini (Peri) are sensitive to proteinase K and are digested. The cytoplasmically oriented carboxyl termini (Cyto) are protected from proteinase K in the absence of CHAPS and digested when CHAPS is added. The DS-LL and 7A-LL mutants are anchored to the membrane in the N-C orientation. The periplasmically oriented carboxyl termini are sensitive to proteinase K and are digested. The cytoplasmically oriented amino termini are protected from proteinase K when no CHAPS is added and digested in the presence of CHAPS. The molecular weight of the protected fragments is larger than the molecular weight markers due to the internally inserted polyleucine segment. The molecular weight markers (TLF and TSF) were loaded in the firstlane. Emptyarrowheads, TLF and the proteinase K-generated large amino-terminal fragments. Filledarrowheads, TSF and the proteinase K-generated small carboxyl fragments. Mutants DS-LL (made without a signal peptide), 2A-LL, and 6A-LL are predominantly in the mature form (m), while mutant 7A-LL retains the signal peptide and is mostly in the precursor (p) form.




DISCUSSION

By converting a normally soluble, periplasmic protein into an artificial membrane protein, the hydrophobicity and length requirements for a segment to stop translocation and anchor the protein to the membrane were examined. For a segment of typical transmembrane length (21 residues), a polymer of contiguous alanine residues was not sufficiently hydrophobic for membrane retention; rather, the protein was transported and retained substantial enzyme activity. In contrast, the incorporation of a polymer of leucines of the same length resulted in stable membrane association and stopped the translocation of the remaining carboxyl-terminal portion of the polypeptide chain. The transition point for stop transfer activity was defined as a segment with a total of 16 alanines and 5 leucines (mutant CN-LA2). In future studies, it will be interesting to determine if the threshold value is influenced by the position of the most highly hydrophobic residues within the transmembrane segment and to examine the extent to which the flanking net charge may modulate this threshold value.

Interestingly, a short segment of only 11 total leucine residues (mutant CN-L) also serves as a stop transfer segment and, as judged by NaOH extraction, is more membrane stable than the longer CN-LA3 mutant. Hydropathy analysis (using scales described in Kyte and Doolittle (1982), Eisenberg et al.(1982), and Engelman et al.(1986)) indicates that the total hydrophobicity of the segments in mutants CN-L and CN-LA2 (11 leucines versus 16 alanines and 5 leucines, respectively) is similar, suggesting that overall hydrophobicity as opposed to length is the critical feature. Nevertheless, few native membrane proteins have such short segments, and this may be because of the unfavorable perturbation of lipid bilayer structure that would be necessary to maintain favorable hydrophobic contacts (Tahara et al., 1992).

Based on theoretical considerations of the energy involved in membrane integration of transmembrane segments, Engelman et al. (1986) suggested that a segment of 20 amino acids with a total transfer energy of 20 kcal/mol (based on the scale described therein) may be the minimum value required for a segment to stop and anchor a protein to the membrane. This is equivalent to about 20 glycine residues and is well below the value for the corresponding polyalanine segment, which did not stop alkaline phosphatase transport. This could reflect a difference between polytopic membrane proteins and single-segment membrane-spanning proteins. Analysis of the amino acid composition of transmembrane segments from these two classes of proteins (von Heijne and Gavel, 1988) is consistent with this notion. Furthermore, the total hydrophobicity of individual transmembrane segments within a polytopic protein, such as the bacterial photoreaction center and bacteriorhodopsin, does vary considerably, and some highly hydrophobic segments may compensate for others that are only weakly so. In addition, these proteins may be further stabilized in the membrane through interactions between transmembrane segments (Khorana et al., 1979; Rees et al., 1989).

In this study, the orientation of the membrane-anchored alkaline phosphatase was found to depend on the export competence of the amino-terminal signal peptide as depicted in the model in Fig. 8. When a functional signal peptide is present, it is cleaved as usual, the internal transmembrane segment functions as a stop transfer sequence, and the protein is anchored in an N-C orientation (mutants 6A-LL, 2A-LL, and CN-LL). However, when the amino-terminal signal peptide is attenuated or deleted, alkaline phosphatase remains in the precursor form, and the internal segment functions as a signal anchor in a C-N orientation (mutants 7A-LL and DS-LL). It is not surprising that in the former case, the functional signal peptide is more favored than the internal segment as a translocation initiation signal since it can engage the transport pathway even before the internal segment is synthesized. In contrast, when the signal peptide is deleted or made dysfunctional, the mutant proteins must initiate translocation at a later stage of translation or do so post-translationally. Regardless, it is remarkable that the internal segment, about 280 amino acids from the amino terminus, directs export and topogenesis. Furthermore, the translocation of these mutants, as well as that of the series with the wild type signal peptide, was inhibited by azide (Oliver et al., 1990), suggesting that they function via SecA, a primary component of the secretory pathway (data not shown).


Figure 8: Model of the orientation of membrane-bound alkaline phosphatase with competent and incompetent (or deleted) signal peptides. A, model for mutants 2A-LL and 6A-LL, which have a functional amino-terminal signal peptide (filledbox). Translocation of mutant proteins is initiated by the amino-terminal signal peptide, which is later cleaved. The translocation is stopped by the internal transmembrane segment (shadedbox), and the protein is anchored to the membrane in an N-C orientation. B, model for mutants with a deleted (DS-LL) or a dysfunctional (7A-LL) amino-terminal signal peptide (emptybox). Since the amino-terminal signal is absent or not functional, translocation of mutant proteins is initiated by the internal transmembrane segment (shadedbox). The carboxyl-terminal polypeptide chain is translocated, and the proteins are anchored to the membrane in a C-N orientation. The amino terminus remains in the cytoplasm (Cyto), and the mutant protein is mainly in the precursor form. Peri, periplasm.



Both statistical and experimental analyses have suggested that the charge of the regions flanking transmembrane segments plays a dominant role in determining the orientation of the transmembrane segment with the more positively charged region remaining cytoplasmic (von Heijne and Gavel, 1988). In this study, the internal transmembrane segment is flanked by a net amino-terminal +2.5 charge and a net carboxyl-terminal +0.5 charge. Nevertheless, the transmembrane segment was able to adopt both N-C and N-C orientations, similar to the observations of Andrews et al.(1992). It is very likely that, in addition to charge, other factors, such as the presence of a cleavable signal peptide, may influence the orientation of a transmembrane segment. Blobel(1980) proposed that the orientation of a transmembrane segment is determined by its relative position with respect to the most amino-terminal signal peptide or signal anchor and that a membrane protein is incorporated into the membrane by the sequential actions of signal anchors and stop-transfer sequences. The orientation of the most amino-terminal signal anchor, or signal peptide, may well be determined by the surrounding positive charges according to the positive inside rule (Hartmann et al., 1989; Lipp et al., 1989), but the orientation of subsequent segments is then influenced by this first transmembrane segment. Our results are consistent with this model. In the presence of a functional signal peptide, protein translocation is initiated by the amino-terminal signal peptide, and the internal transmembrane segment functions as a stop transfer sequence; it stops further translocation and anchors the protein to the membrane in an N-C orientation. When the amino-terminal signal peptide is deleted or dysfunctional, the internal transmembrane segment can initiate protein translocation and function as a signal anchor. The orientation of the transmembrane segment agrees with the positive inside rule, and the protein is anchored to the membrane in a C-N orientation.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grant GM37639 (to D. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Box U-44, The University of Connecticut, Storrs, CT 06269. Tel.: 203-486-1891; Fax: 203-486-1784; E-mail: kendall@uconnvm.uconn.edu.

The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate.


ACKNOWLEDGEMENTS

We thank Linda Randall for sharing her expertise and teaching us flotation gradient centrifugation.


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