Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon

The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.     

Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.     

Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.     

Cancer associated mutations in Sec61γ alter the permeability of the ER translocase

Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.     

Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.     

Ribosome-independent regulation of translocon composition and Sec61alpha conformation

In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61alpha, ribophorin I, and TRAPalpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61alpha, ribophorin I, or TRAPalpha were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61alpha was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61alpha to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61alpha, indicating that a protease-resistant conformation of Sec61alpha can be conferred in a ribosome-independent manner.     

Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein.

Selective degradation of proteins at the endoplasmic reticulum (ER-associated degradation) is thought to proceed largely via the cytosolic ubiquitin-proteasome pathway. Recent data have indicated that the dislocation of short-lived integral-membrane proteins to the cytosolic proteolytic system may require components of the Sec61 translocon. Here we show that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer. In yeast mutants expressing functionally attenuated proteasomes, degradation of a short-lived doubly membrane-spanning protein proceeds rapidly through the N-terminal cytosolic domain of the substrate, but slows down considerably when continued degradation of the molecule requires membrane extraction. Thus, proteasomes engaged in ER degradation can directly process transmembrane proteins through a mechanism in which the dislocation of the substrate and its proteolysis are coupled. We therefore propose that the retrograde transport of short-lived substrates may be driven through the activity of the proteasome.     

Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species

Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin-proteasome system. This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon. It had remained a mystery whether ER-localised soluble proteins are at all capable of re-entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport. In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy-terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy-terminus. With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely. Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin-proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.     

mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells

MicroRNA (miRNA) binds to the 3′-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.     

Sec61β facilitates the maintenance of endoplasmic reticulum homeostasis by associating microtubules

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.     

Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes

The injection of heterologous mRNA into fully grown Xenopus oocytes results not only in the synthesis of the heterologous protein but also in a reciprocal decrease in the synthesis of endogenous proteins. This indicates that injected and endogenous mRNAs compete for some component which is rate-limiting for translation in oocytes. We have attempted to identify this rate-limiting translational component. We find that heterologous and homologous polysomes compete with endogenous mRNAs as effectively as naked mRNA, indicating that polysomes do not contain detectable levels of the rate-limiting factor. In addition, we have used micrococcal nuclease digestion and a mRNA-specific oligonucleotide to destroy the mRNA component of polysomes. The remaining polysome factors, when injected into oocytes, failed to stimulate translation. When several eukaryotic translation initiation factors were injected into oocytes, initiation factor 4A consistently increased general oocyte protein synthesis by about twofold. It is possible that the availability of eIF-4A in oocytes is a key factor in limiting the overall rate of protein synthesis.     

mRNAs encoding polarity and exocytosis factors are cotransported with the cortical endoplasmic reticulum to the incipient bud in Saccharomyces cerevisiae

Polarized growth in the budding yeast Saccharomyces cerevisiae depends upon the asymmetric localization and enrichment of polarity and secretion factors at the membrane prior to budding. We examined how these factors (i.e., Cdc42, Sec4, and Sro7) reach the bud site and found that their respective mRNAs localize to the tip of the incipient bud prior to nuclear division. Asymmetric mRNA localization depends upon factors that facilitate ASH1 mRNA localization (e.g., the 3' untranslated region, She proteins 1 to 5, Puf6, actin cytoskeleton, and a physical association with She2). mRNA placement precedes protein enrichment and subsequent bud emergence, implying that mRNA localization contributes to polarization. Correspondingly, mRNAs encoding proteins which are not asymmetrically distributed (i.e., Snc1, Mso1, Tub1, Pex3, and Oxa1) are not polarized. Finally, mutations which affect cortical endoplasmic reticulum (ER) entry and anchoring in the bud (myo4Delta, sec3Delta, and srp101) also affect asymmetric mRNA localization. Bud-localized mRNAs, including ASH1, were found to cofractionate with ER microsomes in a She2- and Sec3-dependent manner; thus, asymmetric mRNA transport and cortical ER inheritance are connected processes in yeast.     

Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus.

During influenza virus infection, protein synthesis is maintained at high levels and a dramatic switch from cellular to viral protein synthesis occurs despite the presence of high levels of functional cellular mRNAs in the cytoplasm of infected cells (M. G. Katze and R. M. Krug, Mol. Cell. Biol. 4:2198-2206, 1984). To determine the step at which the block in cellular mRNA translation occurs, we compared the polysome association of several representative cellular mRNAs (actin, glyceraldehyde-3-phosphate dehydrogenase, and pHe7 mRNAs) in infected and uninfected HeLa cells. We showed that most of these cellular mRNAs remained polysome associated after influenza viral infection, indicating that the elongation of the proteins encoded by these cellular mRNAs was severely inhibited. Because the polysomes containing these cellular mRNAs did not increase in size but either remained the same size or decreased in size, the initiation step in cellular protein synthesis must also have been defective. Several control experiments established that the cellular mRNAs sedimenting in the polysome region of sucrose gradients were in fact associated with polyribosomes. Most definitively, puromycin treatment of infected cells caused the dissociation of polysomes and the release of cellular, as well as viral, mRNAs from the polysomes, indicating that the cellular mRNAs were associated with polysomes that were capable of forming at least a single peptide bond. A similar analysis was performed with HeLa cells infected by adenovirus, which also dramatically shuts down cellular protein synthesis. Again, it was found that most of the cellular mRNAs, which were translatable in reticulocyte extracts, remained associated with polysomes and that there was a combined initiation-elongation block to cellular protein synthesis. In cells infected by both adenovirus and influenza virus, influenza viral mRNAs were on larger polysomes than were several late adenoviral mRNAs with comparably sized coding regions. In addition, after influenza virus superinfection of cells infected by the adenovirus mutant dl331, a situation in which there is a limitation in the amount of functional initiation factor eIF-2 (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986), influenza viral mRNAs, but not late adenoviral mRNAs, were on polysomes. These results indicate that influenza viral mRNAs are better initiators of translation than are late adenoviral mRNAs.