Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

A novel cytosolic class I antigen-processing pathway for endoplasmic-reticulum-targeted proteins

Proteins bearing an endoplasmic reticulum (ER) leader are inserted into the ER followed by cleavage of the signal peptide. Major histocompatibility complex class I-restricted T-cell epitopes can be generated from these proteins by the proteasome after retrotranslocation into the cytosol. Here, we show that an HLA-A(*)0201-restricted epitope from prostate stem cell antigen contains the cleavage site of the ER signal peptidase. The resulting cleavage products fail to bind to HLA-A(*)0201 and are not recognized by T lymphocytes. As processing of prostate stem cell antigen by signal peptidase occurs immediately after co-translational insertion, the epitope must be processed from polypeptides that have never reached the ER. The processing of this epitope depends on the proteasome and the transporter associated with antigen processing and shows a novel pathway of class I processing that relies on the failure of ER-targeted proteins to reach their target compartment.     

US2, a human cytomegalovirus-encoded type I membrane protein, contains a non-cleavable amino-terminal signal peptide.

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.     

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.     

A zein signal sequence functions as a signal-anchor when fused to maize alcohol dehydrogenase.

A chimeric gene, preZad, was constructed encoding a zein signal sequence fused precisely to the amino terminus of maize alcohol dehydrogenase 1. Translocation and processing of this chimeric preZad protein were assayed in vitro using a rabbit reticulocyte lysate translation system supplemented with canine pancreatic microsomes. PreZad was cotranslationally translocated across the vesicular membranes. Unexpectedly, the signal sequence was not removed although a suitable cleavage site was preserved and presented within the vesicle lumen. Failure to cleave the signal sequence was apparently not due to the lack of a beta-turn near the processing site. When a beta-turn was introduced near the cleavage site through site-directed mutagenesis, no processing was observed. PreZad was not solubilized by alkaline treatment of the microsomes, indicating an integral membrane association. Resistance to proteolysis, in the absence of detergent, indicates that preZad is associated with the membranes in a type II orientation (C-terminus in and N-terminus outside the vesicles). Analysis of truncated versions of preZad showed that it is the uncleaved signal sequence that functions as a signal-anchor. Changing the ratio of net charge flanking the signal sequence to less than 1 (N-terminal:C-terminal) did not alter the type II membrane orientation, as would have been predicted by the 'positive-in rule'. Our results provide additional insight into the role of the passenger protein and signal sequence-flanking regions in recognition of a signal peptidase processing site, and the orientation of insertion of a signal-anchor sequence into the endoplasmic reticulum membrane.     

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.     

Bacterial leader peptidase,a membrane protein without a leader peptide,uses the same export pathway as pre-secretory proteins

Leader peptidase typifies a group of proteins of the plasma membrane of E. coli which span the membrane and are synthesized without a cleaved amino-terminal leader (signal) sequence. The membrane assembly properties of these proteins have not been previously reported. We find that the membrane electrochemical potential is necessary for the insertion of a large domain of leader peptidase across the membrane. In the absence of potential, the peptidase accumulates inside the cell in tight association with the. plasma membrane. Upon restoration of the potential, accumulated peptidase inserts across the membrane, indicating that this insertion is not mechanistically coupled to polypeptide chain growth. The normal, trans-bilayer peptidase and that which accumulates in the absence of potential have different conformations, as shown by the relative resistance of the trans-bilayer enzyme to digestion by trypsin or chymotrypsin in cell lysates. Membrane insertion is accompanied by this conformational change. This assembly reaction has several features predicted by the hypothesis of membrane-triggered folding.     

Two pathways for the degradation of the H2 subunit of the asialoglycoprotein receptor in the endoplasmic reticulum

An intermediate of 35 kD accumulates transiently during ER degradation of the H2 subunit of the asialoglycoprotein receptor; it is derived by an endoproteolytic cleavage in the exoplasmic domain near the transmembrane region. In the presence of cycloheximide all of the precursor H2 is converted to this intermediate, which is degraded only after cycloheximide is removed (Wikstrom, L., and H. F. Lodish. 1991. J. Cell Biol. 113:997-1007). Here we have generated mutants of H2 that do not form the 35-kD fragment, either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes. In transfected cells the kinetics of ER degradation of these mutant proteins are indistinguishable from that of wild-type H2, indicating the existence of a second pathway of ER degradation which does not involve formation of the 35-kD fragment. Degradation of H2 in the ER by this alternative pathway is inhibited by TLCK or TPCK, but neither formation nor degradation of the 35-kD fragment is blocked by these reagents. As determined by NH2-terminal sequencing of the 35-kD fragment, formed either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes, the putative cleavage sites are between small polar, uncharged amino acid residues. Substitution of the residues NH2- or COOH-terminal to the cleavage site by large hydrophobic or charged ones decreased the amount of 35-kD fragment formed and in some cases changed the putative cleavage site. Cleavage can also be affected by amino acid substitutions (e.g., to proline or glycine) which change protein conformation. Therefore, the endoprotease that generates the 35-kD fragment has specificity similar to that of signal peptidase. H2a and H2b are isoforms that differ only by a pentapeptide insertion in the exoplasmic juxtamembrane region of H2a. 100% of H2a is degraded in the ER, but up to 30% of H2b folds properly and matures to the cell surface. The sites of cleavage to form the 35-kD fragment are slightly different in H2a and H2b. Two mutant H2b proteins, with either a glycine or proline substitution at the position of insertion of the pentapeptide in H2a, have metabolic fates similar to that of H2a. These mutations are likely to change the protein conformation in this region. Thus the conformation of the juxtamembrane domain of the H2 protein is important in determining its metabolic fate within the ER.     

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.     

Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.     

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.     

Import of honeybee prepromelittin into the endoplasmic reticulum. Requirements for membrane insertion, processing, and sequestration

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Membrane insertion of prepromelittin, assayed as signal sequence removal by signal peptidase, is not dependent on signal recognition particle and docking protein. However, a previously uncharacterized proteinaceous component of the microsomal membrane is required for completion of membrane transfer of promelittin. Furthermore, membrane insertion of prepromelittin is not coupled to translation. These data suggest the signal sequence, in addition to its role in membrane recognition, has a more general function for membrane insertion, cotranslational import of proteins is not an intrinsic feature of microsomes, and at least in certain cases, proteinaceous membrane components are involved in membrane transfer.     

Regulation of gap junction intercellular communication by the ubiquitin system

Intercellular communication via gap junctions plays a critical role in numerous cellular processes, including the control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are aggregates of intercellular channels that enable adjacent cells in solid tissues to directly exchange ions and small molecules. These channels are formed by a family of integral membrane proteins called connexins, of which the best studied is connexin43. Connexins have a high turnover rate in most tissue types, and degradation of connexins is considered to be a tightly regulated process. Post-translational modification of connexins by ubiquitin is emerging as an important event in the regulation of connexin degradation. Ubiquitination is involved in endoplasmic reticulum-associated degradation of connexins as well as in trafficking of connexins to lysosomes. At both the endoplasmic reticulum and the plasma membrane, ubiquitination of connexins is strongly affected by changes in the extracellular environment. There is increasing evidence that the regulation of connexin ubiquitination might be an important mechanism for rapidly modifying the level of functional gap junctions at the plasma membrane, under both normal and pathological conditions. This review discusses the current knowledge about the regulation of intercellular communication via gap junctions by ubiquitination of connexins.     

Cell-free synthesis and assembly of connexins into functional gap junction membrane channels.

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.     

Effect of amino acid substitutions at the signal peptide cleavage site of the Escherichia coli major outer membrane lipoprotein

The requirement for the glycine residue at the COOH terminus of the signal peptide of the precursor of the major Escherichia coli outer membrane lipoprotein was examined. Using oligonucleotide-directed site-specific mutagenesis, this residue was replaced by residues of increasing side chain size. Substitution by serine had no effect on the modification or processing of the prolipoprotein. Substitution by valine or leucine resulted in the accumulation of the unmodified precursor, whereas threonine substitution resulted in slow lipid modification and no detectable processing of the lipid modified precursor. The results indicate that serine is the upper limit on size for the residue at the cleavage site. Larger residues at this position prevent the action of both the glyceride transferase and signal peptidase II enzymes, indicating that the cleavage site residue plays a role in events prior to proteolytic cleavage. The upper limit on size of the cleavage site residue is similar to that found for exported proteins cleaved by signal peptidase I, as well as eucaryotic exported proteins. The possibility that the cleavage site residue may have a role other than active site recognition by the signal peptidase is discussed.     

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.     

Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate     

Tail-anchored and signal-anchored proteins utilize overlapping pathways during membrane insertion

Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion.