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PubMed1995

A C-terminally-anchored Golgi protein is inserted into the endoplasmic reticulum and then transported to the Golgi apparatus.

Unlike conventional membrane proteins of the secretory pathway, proteins anchored to the cytoplasmic surface of membranes by hydrophobic sequences near their C termini follow a posttranslational, signal recognition particle-independent insertion pathway. Many such C-terminally-anchored proteins have restricted intracellular locations, but it is not known whether these proteins are targeted directly to the membranes in which they will ultimately reside. Here we have analyzed the intracellular sorting of the Golgi protein giantin, which consists of a rod-shaped 376-kDa cytoplasmic domain followed by a hydrophobic C-terminal anchor sequence. Unexpectedly, we find that giantin behaves like a conventional secretory protein in that it inserts into the endoplasmic reticulum (ER) and then is transported to the Golgi. A deletion mutant lacking a portion of the cytoplasmic domain adjacent to the membrane anchor still inserts into the ER but fails to reach the Golgi, even though this mutant has a stable folded structure. These findings suggest that the localization of a C-terminally-anchored Golgi protein involves at least three steps: insertion into the ER membrane, controlled incorporation into transport vesicles, and retention within the Golgi.

Amino Acid Sequence↗
PubMed1995

The carboxyl terminus of the membrane-binding domain of cytochrome b5 spans the bilayer of the endoplasmic reticulum.

Preliminary studies (Vergères, G., and Waskell, L. (1992) J. Biol. Chem. 267, 12583-12591) have suggested that the carboxyl-terminal membrane-binding domain of cytochrome b5 traverses the membrane and that the carboxyl terminus is in the lumen of the endoplasmic reticulum. In order to confirm and extend these studies, additional experiments were conducted. The gene coding for rat cytochrome b5 was transcribed and the resulting mRNA was translated in vitro in a rabbit reticulocyte lysate in the presence of microsomes. The binding and topology of cytochrome b5 were investigated by treating microsomes containing the newly incorporated cytochrome b5 with carboxypeptidase Y and trypsin. Our studies indicate that cytochrome b5 is inserted both co- and post-translationally into microsomes in a topology in which the membrane-binding domain spans the bilayer with its COOH terminus in the lumen. Cytochrome b5 is also incorporated into microsomes pretreated with trypsin in a topology indistinguishable from the one resulting from the insertion of the protein into untreated microsomes, reconfirming that cytochrome b5 does not use the signal recognition particle-dependent translocation machinery. Our results do not allow a distinction to be made between a spontaneous insertion mode or some other trypsin-resistant receptor-mediated mechanism. A role for Pro115 in the middle of the membrane-binding domain of cytochrome b5 was also examined by mutating it to an alanine and subsequently characterizing the ability of the mutant protein to be incorporated into membranes. The mutant protein inserted more slowly in vitro into microsomes as well as into pure lipid bilayers by a factor of 2 to 3.

Amino Acid Sequence↗
PubMed1998

Sec-independent insertion of thylakoid membrane proteins. Analysis of insertion forces and identification of a loop intermediate involving the signal peptide.

A group of membrane proteins are synthesized with cleavable signal sequences but inserted into the thylakoid membrane by an unusual Sec/SRP-independent mechanism. In this report we describe a key intermediate in the insertion of one such protein, photosystem II subunit W (PSII-W). A single mutation in the terminal cleavage site partially blocks processing and leads to the formation of an intermediate-size protein in the thylakoid membrane during chloroplast import assays. This protein is in the form of a loop structure: the N and C termini are exposed on the stromal face, whereas the cleavage site has been translocated into the lumen. In this respect the insertion of this protein resembles that of M13 procoat, which also adopts a loop structure during insertion, and we present preliminary evidence that a similar mechanism is used by another thylakoid protein, PSII-X. However, whereas the negatively charged region of procoat is translocated by an apparently electrophoretic mechanism using the DeltamuH+, the corresponding region of PSII-W is equally acidic but insertion is DeltamuH+ independent. We furthermore show that neutralization of this region has no apparent effect on the insertion process. We propose that a central element in this insertion mechanism is a loop structure whose formation is driven by hydrophobic interactions.

Amino Acid Sequence↗
PubMed1998

Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.

Amino Acid Sequence↗
PubMed2004

Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway.

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.

Adenosine Triphosphatases↗
PubMed2007

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.

Arginine↗
PubMed2002

Nuclear export of 5S rRNA-containing ribonucleoprotein complexes requires CRM1 and the RanGTPase cycle.

In Xenopus oocytes, 5S rRNA is exported out of the nucleus in the context of two ribonucleoprotein complexes (RNPs): complexed with transcription factor IIIA as the 7S RNP or as the 5S RNP with ribosomal protein L5. 5S rRNA-containing RNP export takes place at a slow rate in comparison to that of nuclear export signal-containing proteins and the U1 snRNP. Using oocyte microinjection assays we found that the export of 5S RNPs requires nuclear RanGTP and RanGTP hydrolysis and is leptomycin B-sensitive, indicating the process is mediated by the export receptor CRM1. A novel nuclear export signal motif is characterised in a region of L5 also possessing a nuclear import signal, thus identifying a shuttling domain for this protein. This same motif in L5 is found to be required for interaction with CRM1 in vitro and for export in vivo.

Active Transport, Cell Nucleus↗
PubMed2006

Solution structure of a GAAG tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus.

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U.G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif the conformational stabilities are significantly different to each other It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.

Base Sequence↗
PubMed1986

Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor.

We have found that a soluble activity present in the postribosomal supernatant fraction of Saccharomyces cerevisiae stimulates posttranslational translocation of yeast prepro-alpha-factor across yeast microsomal membranes. Stimulation of translocation is not due to a nonspecific affect on ATP levels. The activity is likely to be due to protein(s) as it is destroyed by N-ethylmaleimide, protease, or heat treatment but not by incubation with RNase. Its apparent sedimentation coefficient is approximately 9.6 S.

Biological Transport, Active↗
PubMed1998

Signal sequence recognition in cotranslational translocation by protein components of the endoplasmic reticulum membrane.

We have investigated the role of membrane proteins and lipids during early phases of the cotranslational insertion of secretory proteins into the translocation channel of the endoplasmic reticulum (ER) membrane. We demonstrate that all steps, including the one during which signal sequence recognition occurs, can be reproduced with purified translocation components in detergent solution, in the absence of bulk lipids or a bilayer. Photocross-linking experiments with native membranes show that upon complete insertion into the channel signal sequences are both precisely positioned with respect to the protein components of the channel and contact lipids. Together, these results indicate that signal sequences are bound to a specific binding site at the interface between the channel and the surrounding lipids, and are recognized ultimately by protein-protein interactions. Our data also suggest that at least some signal sequences reach the binding site by transfer through the interior of the channel.

Animals↗
PubMed2000

Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex.

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.

Animals↗
PubMed2000

Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.

Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

Alkaline Phosphatase↗
PubMed2001

The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome.

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗
PubMed1985

Characterization of the 7S RNA and its gene from halobacteria.

The 7S RNA is an abundant nonribosomal RNA in H. halobium and other halobacteria. A specific 7S RNA gene probe shows high homology to genomic DNA of all halobacteria tested but not to those of several other archaebacteria, eubacteria and eukaryotes. All halobacterial genomes seem to carry a single copy of the 7S RNA gene. The coding region of the 7S RNA gene is highly G+C rich whereas the 5'- and 3'-noncoding regions possess a rather low G+C content. An extended double stranded structure for the 7S RNA is deduced from its nucleotide sequence. The 7S RNA of H. halobium (304 nucleotides) resembles in size and structure the 7S-L RNA from mammalian cells and shares with it a sequence homology of about 50% when arranged in a colinear fashion. The similarities in sequence are found particularly at the 3'- and 5'-termini. No similarity was detected between the 7S RNA from H. halobium and the nonribosomal 6S RNA from Escherichia coli.

Animals↗