Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p

Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible for sorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPase demonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and is sufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary and sufficient for trafficking of the V-ATPase within the Golgi/endosomal network.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

Analysis of the signal for attachment of a glycophospholipid membrane anchor

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH-terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane- spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.     

Plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers

A detailed molecular dynamics study of the haemagglutinin fusion peptide (N-terminal 20 residues of the HA2 subunits) in a model bilayer has yielded useful information about the molecular interactions leading to insertion into the lipids. Simulations were performed on the native sequence, as well as a number of mutant sequences, which are either fusogenic or nonfusogenic. For the native sequence and fusogenic mutants, the N-terminal 11 residues of the fusion peptides are helical and insert with a tilt angle of approximately 30 degrees with respect to the membrane normal, in very good agreement with experimental data. The tilted insertion of the native sequence peptide leads to membrane bilayer thinning and the calculated order parameters show larger disorder of the alkyl chains. These results indicate that the lipid packing is perturbed by the fusion peptide and could be used to explain membrane fusion. For the nonfusogenic sequences investigated, it was found that most of them equilibrate parallel to the interface plane and do not adopt a tilted conformation. The presence of a charged residue at the beginning of the sequence (G1E mutant) resulted in a more difficult case, and the outcomes do not fall straightforwardly into the general picture. Sequence searches have revealed similarities of the fusion peptide of influenza haemagglutinin with peptide sequences such as segments of porin, amyloid alpha eta peptide, and a peptide from the prion sequence. These results confirm that the sequence can adopt different folds in different environments. The plasticity and the conformational dependence on the local environment could be used to better understand the function of fusion peptides.     

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.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

An internally positioned signal can direct attachment of a glycophospholipid membrane anchor

All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.     

The role of the N region in signal sequence and signal-anchor function.

To determine the role of sequences other than the hydrophobic core in mediating signal sequence function, we examined the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that neither the N nor the C region of the preprolactin signal sequence is necessary for translocation. However, insertion of sequences with either a net charge of +2.5 or -6.0 between the N region and the hydrophobic core of the signal converted it into a signal-anchor. The topologies adopted (types I and II, respectively) were opposite those predicted from the distribution of charges surrounding the hydrophobic core of the signals. When these mutant signals were located in the interior of an otherwise secreted protein, both sequences functioned as stop-transfer sequences. Related mutations were assayed in fusion proteins in which the IgM transmembrane domain functioned as an amino-terminal signal-anchor. For these molecules, the distribution of charged residues surrounding the hydrophobic core had no influence on the topology adopted. Our results suggest that features other than simple charge distribution play an important role in determining membrane topology in vitro.     

A stop transfer sequence recognizes receptors for nascent chain translocation across the endoplasmic reticulum membrane

A stop transfer sequence derived from the extreme carboxyl terminus of membrane IgM heavy chain has been shown to confer predictable transmembrane orientation to secretory proteins by aborting translocation of subsequently synthesized protein domains. Here we demonstrate that, in certain peptide sequence contexts, the same stop transfer sequence is also capable of initiating domain translocation across the endoplasmic reticulum (ER) membrane. Translocation directed by a stop transfer sequence is similar to, but distinguishable from, the action of a conventional signal sequence. Translocation is dependent on participation of the ribosome and protein receptors both in the cytoplasm and in the ER membrane. Moreover, both amino- and carboxy-terminal flanking protein domains can be translocated. Unlike a signal sequence, the stop transfer sequence is not itself translocated across the membrane. These results have implications for the nature of signal sequences, stop transfer sequences, and their receptor interactions.     

A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export

Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so- called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions -1 and 2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.     

Structural determinants for signal sequence function in the mammalian endoplasmic reticulum

Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.     

Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane

Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein.     

In vitro kinetic analysis of the role of the positive charge at the amino-terminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in Escherichia coli

By using an in vitro system for the translocation of secretory proteins in Escherichia coli, detailed and quantitative studies were performed as to the function of the positively charged amino acid residues at the amino terminus of the signal peptide. Uncleavable OmpF-Lpp, a model secretory protein carrying an uncleavable signal peptide, and mutant proteins derived from it were used as translocation substrates. When the positive charge, +2 (LysArg) for the wild-type, was changed to 0, -1, or -2, little or no translocation was observed. The number of the positive charge was altered by introducing different numbers of Lys or Arg residues into the amino terminus. The rate of translocation was roughly proportional to this number, irrespective of whether the charged amino acid residues were Lys or Arg. When the amino-terminal LysArg was replaced by His residues, translocation took place more efficiently at pH 6.5 than pH 8.0, whereas that of the wild-type was about the same as the two pH values. We conclude that the signal peptide requires a positive charge at its amino-terminal region to function in the translocation reaction and that the rate of translocation is roughly proportional to the number of the positively charged group, irrespective of the amino acid species that donates the charge. Evidence suggesting that the positive charge is involved in the binding of precursor proteins to the membrane surface to initiate translocation is also presented.     

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).     

Sequences beyond the cleavage site influence signal peptide function

The earliest events in protein secretion include targeting to and translocation across the endoplasmic reticulum membrane. To dissect the mechanism by which signal sequences mediate translocation in eukaryotes, we are examining the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that the protein domain being translocated can have profound impact on the efficiency of the translocation process. Specifically, deletions in the mature prolactin "passenger" domain, beyond the signal cleavage site, reduce the efficiency of signal function. The effect of these deletions on signal function is observed when this signal sequence is in its normal position, at the amino terminus, and when internalized by the addition of 117 amino acids of chimpanzee alpha-globin. Alterations in the interaction of the deletion mutants with the signal recognition particle and with another component of the translocation system, signal peptidase, were observed. Our results suggest that subtle changes in sequences beyond the signal cleavage site can alter the efficiency of co-translational translocation by affecting various signal-receptor interactions.     

Sequences in rotavirus glycoprotein VP7 that mediate delayed translocation and retention of the protein in the endoplasmic reticulum

Glycosylation and translocation of the simian rotavirus protein VP7, a resident ER protein, does not occur co-translationally in vivo. In pulse-chase experiments in COS cells, nonglycosylated VP7 was still detectable after a 25-min chase period, although the single glycosylation site was only 18 residues beyond the signal peptide cleavage site. After labeling, glycosylated and nonglycosylated VP7 was recovered in microsomes but the latter was sensitive to trypsin (i.e., the nascent protein became membrane associated) but most of it entered the ER posttranslationally because of a rate-limiting step early in translocation. In contrast with the simian protein, bovine VP7 was glycosylated and translocated rapidly. Thus, delayed translocation per se was not required for retention of VP7 in the ER. By constructing hybrid proteins, it was further shown that the signal peptide together with residues 64-111 of the simian protein caused delayed translocation. The same sequences were also necessary and sufficient for retention of simian VP7 in the ER. The data are consistent with the idea that certain proteins are inserted into the ER membrane in a loop configuration.