Sec61p contributes to signal sequence orientation according to the positive-inside rule

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the "positive-inside rule." We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.     

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.     

Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion

Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.     

Manipulation of membrane protein topology on the endoplasmic reticulum by a specific ligand in living cells

Almost all integral membrane proteins in the secretory pathway are cotranslationally inserted into the endoplasmic reticulum membrane. Their membrane topology is determined by their amino acid sequences. Here we show that the topology can be manipulated by a factor other than the amino acid sequence. A dihydrofolate reductase (DHFR) domain was fused to the N-terminus of the type I signal-anchor sequence of synaptotagmin II, which mediates translocation of the preceding portion. The DHFR domain was translocated through the membrane in COS7 cells and a transmembrane (TM) topology was achieved. When a DHFR ligand, methotrexate, was added to the culture medium, translocation of the DHFR domain was suppressed and both ends of the signal-anchor sequence remained on the cytoplasmic side. In contrast, translocation of the DHFR domain fused after the signal peptide, which translocates the following region, was not affected by the ligand. The topology-altered fusion protein was anchored to the membrane in a high salt-resistant state, and partially extracted from the membrane under alkali conditions. We concluded that the topology of membrane proteins can be manipulated by a trans-acting factor, even in living cells.     

Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N(exoplasmic)/C(cytoplasmic) topology. This topology results in the translocation of the smallest ectodomain ( approximately 20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.     

Elements in the N-terminal signaling sequence that determine cytosolic topology of short-chain dehydrogenases/reductases. Studies with retinol dehydrogenase type 1 and cis-retinol/androgen dehydrogenase type 1

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11beta-hydroxysteroid dehydrogenase (11beta-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11beta-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.     

Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence

We have investigated the topogenic rules of multispanning membrane proteins using erythrocyte band 3. Here, the fine structural requirements for the correct disposition of its second transmembrane segment (TM2) were assessed. We made fusion proteins where TM1 and the loop sequence preceding TM2 were changed and fused to prolactin. They were expressed in a cell-free system supplemented with rough microsomal membrane, and their topologies on the membrane were assessed by protease sensitivity and N-glycosylation. TM1 was demonstrated to be a signal-anchor sequence that mediates translocation of the downstream portion, and thus TM2 should be responsible to halt the translocation to acquire TM topology. When the loop between TM1 and TM2 was elongated, however, TM2 was readily translocated through the membrane and not integrated. For the membrane integration of TM2, TM2 must be in close proximity to TM1. The TM1 can be replaced with another signal-anchor sequence with a long hydrophobic segment but not with a signal sequence with shorter hydrophobic stretch. The length of the hydrophobic segment affected final topology of TM2. We concluded that the two TM segments work synergistically within the translocon to acquire the correct topology and that the length of the preceding signal sequence is critical for stable transmembrane assembly of TM2. We propose that direct interaction among the TM segments is one of the critical factors for the transmembrane topogenesis of multispanning membrane proteins.     

Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT

I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.     

Secretion of FGF-16 requires an uncleaved bipartite signal sequence

Fibroblast growth factor (FGF)-16 is one of the rare secreted proteins that do not possess a cleavable signal sequence. Here we describe our examination of the mechanism and structural requirements for the secretion of FGF-16 from COS-1 transfectants. Inhibition of its secretion by brefeldin A and identification of an N-glycan on the secreted form confirmed that FGF-16 is secreted by means of the endoplasmic reticulum and Golgi apparatus, as are secreted proteins having a conventional cleavable signal sequence. Deletion of its N terminus abolished secretion of FGF-16. When chimerized with prolactin, however, the N-terminal sequence of FGF-16 was not able to mediate secretion of the chimera. Point mutations that made the N terminus less hydrophobic had little effect on secretion of FGF-16, whereas making the central hydrophobic region less hydrophobic abolished secretion. Within cells, an unsecretable FGF-16 N-terminal deletion mutant was distributed in the perinuclear region and overlapped the distribution of the Golgi apparatus. Mutants with less hydrophobic central regions were distributed evenly throughout the cytosol. Collectively, these results indicate that FGF-16 employs a unique bipartite signal sequence (i.e. both the N-terminal region and central hydrophobic region) that is not cleaved, although it shares the same secretory machinery used by secreted proteins with cleavable signal sequences.     

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.     

Topogenesis of two transmembrane type K+ channels,Kir 2.1 and KcsA

Potassium channels, which control the passage of K+ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K+ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K+ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the Ncyt/Cexo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signal-anchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.     

Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.     

Redundancy of signal and anchor functions in the NH2-terminal uncharged region of influenza virus neuraminidase, a class II membrane glycoprotein

Class II membrane glycoproteins share a common topology of the NH2 terminus inside and the COOH terminus outside the cell. Their transport to the cell surface is initiated by the function of a single hydrophobic domain near the NH2 terminus. This functional domain serves both as an uncleaved signal sequence and as a transmembrane anchor. We examined the signal and anchor functions of influenza virus neuraminidase, a prototype class II membrane glycoprotein, by deletion analysis of its long, uncharged amino-terminal region. The results presented here show that the entire stretch of 29 uncharged amino acids (7 to 35) is not required for either a signal sequence or an anchor sequence function. On the basis of translocation and membrane stability data for different mutants, we suggest that the first 20 amino acid residues (7 to 27) are likely to provide the hydrophobic core for these functions and that within this putative subdomain some sequences are more efficient than the other sequences in providing a translocation function. Finally, it appears that neuraminidase and its mutant proteins are translocated with the proper orientation, regardless of the characteristics of the flanking sequences.     

Features of transmembrane segments that promote the lateral release from the translocase into the lipid phase

Topogenic sequences direct the membrane topology of proteins by being recognized and decoded by integral membrane translocases. In this paper, we have compared the minimal sequence characteristics of helical-hairpin, reverse signal-anchor, and stop-transfer sequences in bacterial membrane proteins that use either the YidC or SecYEG translocases for membrane insertion. We find that a stretch composed of 3 leucines and 16 alanines is required for efficient membrane-anchoring of the M13 procoat protein that inserts by a helical hairpin mechanism, and that a stretch composed of only 19 alanines has a detectable membrane-anchoring ability. Similar results were obtained for the reverse signal-anchor sequence of the single-spanning Pf3 coat protein and for stop-transfer segments engineered into leader peptidase. We have also determined the contribution to the apparent free energy of membrane insertion of M13 procoat for all 20 amino acids. The relative order of the contributions is similar to that determined for a stop-transfer sequence in the mammalian endoplasmic reticulum, but the absolute difference between the contributions for the most hydrophobic and most hydrophilic residues is somewhat larger in the E. coli system. These results are significant because they define the features of a membrane protein transmembrane segment that induce lateral release from the YidC and Sec translocases into the lipid bilayer in bacteria.     

Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors

The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an α-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide's affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.