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PubMed1999

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae.

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

Alleles↗

PubMed2000

Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex.

SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex. We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex. The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p. The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition.

Amino Acid Sequence↗

PubMed2001

The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p.

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

Acyltransferases↗

PubMed1998

Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins.

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele. This is accompanied by the stabilization of the Sec61-2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.

Amino Acid Sequence↗

PubMed2002

Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein.

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.

Animals↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2005

The cotranslational maturation of the type I membrane glycoprotein tyrosinase: the heat shock protein 70 system hands off to the lectin-based chaperone system.

The maturation of eukaryotic secretory cargo initiates cotranslationally and cotranslocationally as the polypeptide chain emerges into the endoplasmic reticulum lumen. Here, we characterized the cotranslational maturation pathway for the human type I membrane glycoprotein tyrosinase. To recapitulate the cotranslational events, including glycosylation, signal sequence cleavage, chaperone binding, and oxidation, abbreviated transcripts lacking a stop codon were in vitro translated in the presence of semipermeabilized melanocyte membranes. This created a series of ribosome/translocon-arrested chains of increasing lengths, simulating intermediates in the cotranslational folding process. Initially, nascent chains were found to associate with the heat shock protein (Hsp) 70 family member BiP. As the nascent chains elongated and additional glycans were transferred, BiP binding rapidly decreased and the lectin-based chaperone system was recruited in its place. The lectin chaperone calnexin bound to the nascent chain after the addition of two glycans, and calreticulin association followed upon the addition of a third. The glycan-specific oxidoreductase ERp57 was cross-linked to tyrosinase when calnexin and calreticulin were associated. This timing coincided with the formation of disulfide bonds within tyrosinase and the cleavage of its signal sequence. Therefore, tyrosinase maturation initiates cotranslationally with the Hsp70 system and is handed off to the lectin chaperone system that first uses calnexin before calreticulin. Interestingly, divergence in the maturation pathways of wild-type and mutant albino tyrosinase can already be observed for translocon-arrested nascent chains.

Animals↗

PubMed2006

EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol.

The plant toxin ricin is transported retrogradely from the cell surface to the endoplasmic reticulum (ER) from where the enzymatically active part is retrotranslocated to the cytosol, presumably by the same mechanism as used by misfolded proteins. The ER degradation enhancing alpha-mannosidase I-like protein, EDEM, is responsible for directing aberrant proteins for ER-associated protein degradation. In this study, we have investigated whether EDEM is involved in ricin retrotranslocation. Overexpression of EDEM strongly protects against ricin. However, when the interaction between EDEM and misfolded proteins is inhibited by kifunensin, EDEM promotes retrotranslocation of ricin from the ER to the cytosol. Furthermore, puromycin, which inhibits synthesis and thereby transport of proteins into the ER, counteracted the protection seen in EDEM-transfected cells. Coimmunoprecipitation studies revealed that ricin can interact with EDEM and with Sec61alpha, and both kifunensin and puromycin increase these interactions. Importantly, vector-based RNA interference against EDEM, which leads to reduction of the cellular level of EDEM, decreased retrotranslocation of ricin A-chain to the cytosol. In conclusion, our results indicate that EDEM is involved in retrotranslocation of ricin from the ER to the cytosol.

Alkaloids↗

PubMed2006

Rtn1p is involved in structuring the cortical endoplasmic reticulum.

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

Endoplasmic Reticulum↗

PubMed2007

Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression.

The epidermal growth factor (EGF)-dependent trafficking of the intact EGF receptor to the nucleus and its requirement for growth factor induction of cyclin D and other genes has been reported. Unresolved is the mechanism by which this or other transmembrane proteins are excised from a lipid bilayer before nuclear translocalization. We report that, after the addition of EGF, the cell surface EGF receptor is trafficked to the endoplasmic reticulum (ER) where it associates with Sec61beta, a component of the Sec61 translocon, and is retrotranslocated from the ER to the cytoplasm. Abrogation of Sec61beta expression prevents EGF-dependent localization of EGF receptors to the nucleus and expression of cyclin D. This indicates that EGF receptors are trafficked from the ER to the nucleus by a novel pathway that involves the Sec61 translocon.

Animals↗

PubMed1994

Cloning and characterization of the ribosomal protein genes in the spc operon of a prokaryotic endosymbiont of the pea aphid, Acyrthosiphon kondoi.

To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphon kondoi, with the endosymbionts in related aphid species as well as with free-living bacteria and subcellular organelles, and to study the mode of its gene expression within aphid cells, we have cloned and characterized the genes encoding ribosomal proteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18, S5, L30, L15 and secretion protein Y (Sec Y) from the S10 and spc ribosomal protein gene operons of this endosymbiont. The organization of these genes is identical to that in Escherichia coli, and their nucleotide sequences are highly similar (87% identity) to the corresponding E. coli genes. They are much less similar to the corresponding chloroplast and mitochondrial genes. The guanine plus cytosine G+C content of the genes of the A. kondoi endosymbiont is much higher than those of the endosymbionts in related aphid species reported so far. It appears either that the A. kondoi endosymbiont is derived from an ancestral bacterium different from those in other aphids or that its G+C content increased in a relatively short time after the evolutionary divergence of its host.

Amino Acid Sequence↗

PubMed2001

Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast.

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.

ATP-Binding Cassette Transporters↗

PubMed2001

Reconstitution of Sec-dependent membrane protein insertion: nascent FtsQ interacts with YidC in a SecYEG-dependent manner.

The inner membrane protein YidC is associated with the preprotein translocase of Escherichia coli and contacts transmembrane segments of nascent inner membrane proteins during membrane insertion. YidC was purified to homogeneity and co-reconstituted with the SecYEG complex. YidC had no effect on the SecA/SecYEG-mediated translocation of the secretory protein proOmpA; however, using a crosslinking approach, the transmembrane segment of nascent FtsQ was found to gain access to YidC via SecY. These data indicate the functional reconstitution of the initial stages of YidC-dependent membrane protein insertion via the SecYEG complex.

Adenosine Triphosphatases↗

PubMed2001

Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC.

Recent studies identified YidC as a novel membrane factor that may play a key role in membrane insertion of inner membrane proteins (IMPs), both in conjunction with the Sec-translocase and as a separate entity. Here, we show that the type II IMP FtsQ requires both the translocase and, to a lesser extent, YidC in vivo. Using photo-crosslinking we demonstrate that the transmembrane (TM) domain of the nascent IMP FtsQ inserts into the membrane close to SecY and lipids, and moves to a combined YidC/lipid environment upon elongation. These data are consistent with a crucial role for YidC in the lateral transfer of TM domains from the Sec translocase into the lipid bilayer.

Adenosine Triphosphatases↗

PubMed1997

Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme.

Escherichia coli preprotein translocase contains a membrane-embedded trimeric complex of SecY, SecE and SecG (SecYEG) and the peripheral SecA protein. SecYE is the conserved functional 'core' of the SecYEG complex. Although sufficient to provide sites for high-affinity binding and membrane insertion of SecA, and for its activation as a preprotein-dependent ATPase, SecYE has only very low capacity to support translocation. The proteins encoded by the secD operon--SecD, SecF and YajC--also form an integral membrane heterotrimeric complex (SecDFyajC). Physical and functional studies show that these two trimeric complexes are associated to form SecYEGDFyajC, the hexameric integral membrane domain of the preprotein translocase 'holoenzyme'. Either SecG or SecDFyajC can support the translocation activity of SecYE by facilitating the ATP-driven cycle of SecA membrane insertion and de-insertion at different stages of the translocation reaction. Our findings show that each of the prokaryote-specific subunits (SecA, SecG and SecDFyajC) function together to promote preprotein movement at the SecYE core of the translocase.

Adenosine Triphosphatases↗

PubMed1997

Molecular architecture of the ER translocase probed by chemical crosslinking of Sss1p to complementary fragments of Sec61p.

The heterotrimeric Sec61p complex is a key component of the protein translocation apparatus of the endoplasmic reticulum membrane. The complex characterized from yeast includes Sec61p, a 10-transmembrane-domain membrane protein which has a direct interaction with Sss1p, a small C-terminal anchor protein. In order to gain some insight into the architecture of this complex we have functionally expressed Sec61p as complementary N- and C-terminal fragments. Chemical crosslinking of Sss1p to specific Sec61p fragments in these functional combinations and suppression of sec61 mutants by over-expression of Sss1p have led to identification of the region which includes transmembrane domains TM6, TM7 and TM8 (amino acid residues L232-R406) of Sec61p as a major site of interaction with Sss1p.

Amino Acid Sequence↗

PubMed1997

The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling.

Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein.

Adenosine Triphosphatases↗

PubMed1997

The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.

The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.

Adenosine Triphosphatases↗