FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3048449614> ?p ?o ?g. }

• W3048449614 endingPage "1849" @default.
• W3048449614 startingPage "1826" @default.
• W3048449614 abstract "Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation. Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation. RNA localization and accompanying local translation serve critical roles in the spatiotemporal regulation of post-transcriptional gene expression. Reflecting the importance of such regulation, localized mRNA translation requires the coordinate localization of numerous proteins, including aminoacyl-tRNA synthetases, translation factors, RNA-binding proteins (RBPs), molecular chaperones, enzymes/scaffolding proteins which act to modify the nascent polypeptide chain, as well as cis-encoded mRNA localization and trafficking information (1Gunkel N. Yano T. Markussen F.H. Olsen L.C. Ephrussi A. Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA.Genes Dev. 1998; 12: 1652-1664Crossref PubMed Google Scholar, 2Smibert C.A. Lie Y.S. Shillinglaw W. Henzel W.J. Macdonald P.M. Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro.RNA. 1999; 5: 1535-1547Crossref PubMed Scopus (108) Google Scholar, 3Micklem D.R. Adams J. Grunert S. St Johnston D. Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation.EMBO J. 2000; 19: 1366-1377Crossref PubMed Google Scholar, 4Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons.J. Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar, 5Gu W. Deng Y. Zenklusen D. Singer R.H. A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization.Genes Dev. 2004; 18: 1452-1465Crossref PubMed Scopus (139) Google Scholar, 6Huttelmaier S. Zenklusen D. Lederer M. Dictenberg J. Lorenz M. Meng X. Bassell G.J. Condeelis J. Singer R.H. Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1.Nature. 2005; 438: 512-515Crossref PubMed Scopus (452) Google Scholar, 7Paquin N. Menade M. Poirier G. Donato D. Drouet E. Chartrand P. Local activation of yeast ASH1 mRNA translation through phosphorylation of Khd1p by the casein kinase Yck1p.Mol. Cell. 2007; 26: 795-809Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 8Willett M. Brocard M. Davide A. Morley S.J. Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts.Biochem. J. 2011; 438: 217-227Crossref PubMed Scopus (25) Google Scholar, 9Yasuda K. Kotani T. Yamashita M. A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing.Developmental Biol. 2013; 382: 517-529Crossref PubMed Scopus (12) Google Scholar, 10Zhang Q. Meng X. Li D. Chen S. Luo J. Zhu L. Singer R.H. Gu W. Binding of DEAD-box helicase Dhh1 to the 5‘-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.J. Biol. Chem. 2017; 292: 9787-9800Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 11Bellon A. Iyer A. Bridi S. Lee F.C.Y. Ovando-Vazquez C. Corradi E. Longhi S. Roccuzzo M. Strohbuecker S. Naik S. Sarkies P. Miska E. Abreu-Goodger C. Holt C.E. Baudet M.L. miR-182 regulates Slit2-mediated axon guidance by modulating the local translation of a specific mRNA.Cell Reports. 2017; 18: 1171-1186Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 12Vidaki M. Drees F. Saxena T. Lanslots E. Taliaferro M.J. Tatarakis A. Burge C.B. Wang E.T. Gertler F.B. A requirement for Mena, an actin regulator, in local mRNA translation in developing neurons.Neuron. 2017; 95: 608-622.e5Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 13Debard S. Bader G. De Craene J.O. Enkler L. Bar S. Laporte D. Hammann P. Myslinski E. Senger B. Friant S. Becker H.D. Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.Methods. 2017; 113: 91-104Crossref PubMed Scopus (12) Google Scholar, 14Koppers M. Cagnetta R. Shigeoka T. Wunderlich L.C. Vallejo-Ramirez P. Qiaojin Lin J. Zhao S. Jakobs M.A. Dwivedy A. Minett M.S. Bellon A. Kaminski C.F. Harris W.A. Flanagan J.G. Holt C.E. Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons.eLife. 2019; 8Crossref PubMed Scopus (10) Google Scholar). At the endoplasmic reticulum (ER), the primary site for secretory and membrane protein synthesis, mRNA translation becomes more complex, requiring additional protein factors including proteins that facilitate ribosome association with the ER membrane, namely the protein translocation machinery, which participates in ribosome association, and newly discovered non-canonical integral membrane RNA-binding proteins (15Beckmann R. Spahn C.M. Eswar N. Helmers J. Penczek P.A. Sali A. Frank J. Blobel G. Architecture of the protein-conducting channel associated with the translating 80S ribosome.Cell. 2001; 107: 361-372Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 16Berkovits B.D. Mayr C. Alternative 3‘ UTRs act as scaffolds to regulate membrane protein localization.Nature. 2015; 522: 363-367Crossref PubMed Scopus (0) Google Scholar, 17Cui X.A. Zhang H. Palazzo A.F. p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.PLoS Biol. 2012; 10: e1001336Crossref PubMed Scopus (66) Google Scholar, 18Gorlich D. Prehn S. Hartmann E. Kalies K.U. Rapoport T.A. A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation.Cell. 1992; 71: 489-503Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 19Hsu J.C. Reid D.W. Hoffman A.M. Sarkar D. Nicchitta C.V. Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.RNA. 2018; 24: 688-703Crossref PubMed Scopus (10) Google Scholar, 20Jagannathan S. Hsu J.C. Reid D.W. Chen Q. Thompson W.J. Moseley A.M. Nicchitta C.V. Multifunctional Roles for the Protein Translocation Machinery in RNA Anchoring to the Endoplasmic Reticulum.J. Biol. Chem. 2014; 289: 25907-25924Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 21Jan C.H. Williams C.C. Weissman J.S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2014; 346: 1257521Crossref PubMed Scopus (168) Google Scholar, 22Johnson A.E. van Waes M.A. The translocon: a dynamic gateway at the ER membrane.Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (479) Google Scholar, 23Rapoport T.A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.Nature. 2007; 450: 663-669Crossref PubMed Scopus (612) Google Scholar, 24Reid D.W. Nicchitta C.V. Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling.J. Biol. Chem. 2012; 287: 5518-5527Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25Reid D.W. Nicchitta C.V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2015; 16: 221-231Crossref PubMed Scopus (85) Google Scholar, 26Simsek D. Tiu G.C. Flynn R.A. Byeon G.W. Leppek K. Xu A.F. Chang H.Y. Barna M. The mammalian Ribo-interactome reveals ribosome functional diversity and heterogeneity.Cell. 2017; 169: 1051-1065.e1018Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27Stephens S.B. Dodd R.D. Brewer J.W. Lager P.J. Keene J.D. Nicchitta C.V. Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation.Mol. Biol. Cell. 2005; 16: 5819-5831Crossref PubMed Scopus (66) Google Scholar, 28Voigt F. Zhang H. Cui X.A. Triebold D. Liu A.X. Eglinger J. Lee E.S. Chao J.A. Palazzo A.F. Single-molecule quantification of translation-dependent association of mRNAs with the endoplasmic reticulum.Cell Reports. 2017; 21: 3740-3753Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 29Walter P. Blobel G. Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in vitro-assembled polysomes synthesizing secretory proteins.J. Cell Biol. 1981; 91: 551-556Crossref PubMed Google Scholar, 30Walter P. Blobel G. Translocation of proteins across the endoplasmic reticulum. III. Signal recognition protein (SRP) causes signal sequence dependent and site-specific arrest of chain elongation that is released by microsomal membranes.J. Cell Biol. 1981; 91: 557-561Crossref PubMed Google Scholar). With understanding of the structural organization and regulation of ER-associated translation being largely derived from the classical canine pancreas rough microsome system, a largely unexplored question in the field concerns the cellular components and mechanisms regulating ER-localized translation in intact cells. A further level of complexity to ER-localized protein synthesis appears when considering the multiple lines of evidence supporting a transcriptome-wide role for the ER in proteome expression (17Cui X.A. Zhang H. Palazzo A.F. p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.PLoS Biol. 2012; 10: e1001336Crossref PubMed Scopus (66) Google Scholar, 21Jan C.H. Williams C.C. Weissman J.S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2014; 346: 1257521Crossref PubMed Scopus (168) Google Scholar, 24Reid D.W. Nicchitta C.V. Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling.J. Biol. Chem. 2012; 287: 5518-5527Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25Reid D.W. Nicchitta C.V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2015; 16: 221-231Crossref PubMed Scopus (85) Google Scholar, 28Voigt F. Zhang H. Cui X.A. Triebold D. Liu A.X. Eglinger J. Lee E.S. Chao J.A. Palazzo A.F. Single-molecule quantification of translation-dependent association of mRNAs with the endoplasmic reticulum.Cell Reports. 2017; 21: 3740-3753Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 31Chartron J.W. Hunt K.C. Frydman J. Cotranslational signal-independent SRP preloading during membrane targeting.Nature. 2016; 536: 224-228Crossref PubMed Scopus (65) Google Scholar, 32Diehn M. Bhattacharya R. Botstein D. Brown P.O. Genome-scale identification of membrane-associated human mRNAs.PLoS Genet. 2006; 2: e11Crossref PubMed Scopus (60) Google Scholar, 33Diehn M. Eisen M.B. Botstein D. Brown P.O. Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.Nat. Genet. 2000; 25: 58-62Crossref PubMed Scopus (178) Google Scholar, 34Hoffman A.M. Chen Q. Zheng T. Nicchitta C.V. Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis.J. Biol. Chem. 2019; 294: 8942-8958Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 35Lerner R.S. Seiser R.M. Zheng T. Lager P.J. Reedy M.C. Keene J.D. Nicchitta C.V. Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes.Rna. 2003; 9: 1123-1137Crossref PubMed Scopus (105) Google Scholar, 36Mueckler M.M. Pitot H.C. Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations.J. Cell Biol. 1981; 90: 495-506Crossref PubMed Google Scholar, 37Mueckler M.M. Pitot H.C. Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles.J. Cell Biol. 1982; 94: 297-307Crossref PubMed Google Scholar). Notably, investigations of ER-localized mRNA composition in human cells, tissues, yeast, and fly revealed that all transcripts, not just those encoding secretory and membrane proteins, are translated on the ER (17Cui X.A. Zhang H. Palazzo A.F. p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.PLoS Biol. 2012; 10: e1001336Crossref PubMed Scopus (66) Google Scholar, 21Jan C.H. Williams C.C. Weissman J.S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2014; 346: 1257521Crossref PubMed Scopus (168) Google Scholar, 24Reid D.W. Nicchitta C.V. Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling.J. Biol. Chem. 2012; 287: 5518-5527Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25Reid D.W. Nicchitta C.V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2015; 16: 221-231Crossref PubMed Scopus (85) Google Scholar, 28Voigt F. Zhang H. Cui X.A. Triebold D. Liu A.X. Eglinger J. Lee E.S. Chao J.A. Palazzo A.F. Single-molecule quantification of translation-dependent association of mRNAs with the endoplasmic reticulum.Cell Reports. 2017; 21: 3740-3753Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 31Chartron J.W. Hunt K.C. Frydman J. Cotranslational signal-independent SRP preloading during membrane targeting.Nature. 2016; 536: 224-228Crossref PubMed Scopus (65) Google Scholar, 33Diehn M. Eisen M.B. Botstein D. Brown P.O. Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.Nat. Genet. 2000; 25: 58-62Crossref PubMed Scopus (178) Google Scholar, 35Lerner R.S. Seiser R.M. Zheng T. Lager P.J. Reedy M.C. Keene J.D. Nicchitta C.V. Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes.Rna. 2003; 9: 1123-1137Crossref PubMed Scopus (105) Google Scholar, 36Mueckler M.M. Pitot H.C. Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations.J. Cell Biol. 1981; 90: 495-506Crossref PubMed Google Scholar, 37Mueckler M.M. Pitot H.C. Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles.J. Cell Biol. 1982; 94: 297-307Crossref PubMed Google Scholar, 38Chen Q. Jagannathan S. Reid D.W. Zheng T. Nicchitta C.V. Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells.Mol. Biol. Cell. 2011; 22: 2646-2658Crossref PubMed Scopus (46) Google Scholar, 39Kopczynski C.C. Noordermeer J.N. Serano T.L. Chen W.Y. Pendleton J.D. Lewis S. Goodman C.S. Rubin G.M. A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis.Proc. Natl. Acad. Sci. U S A. 1998; 95: 9973-9978Crossref PubMed Scopus (93) Google Scholar). Although landmark biochemical and structural studies have advanced our understanding of how secretory/membrane protein synthesis is coupled to protein translocation, it remains unclear how translation on the ER is compartmentalized to accommodate the coincident translation of both cytosolic and secretory/membrane protein-encoding mRNAs. One model proposes that an mRNA-wide role for the ER in proteome expression is achieved by translocon-independent modes of ribosome association with the ER membrane (25Reid D.W. Nicchitta C.V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2015; 16: 221-231Crossref PubMed Scopus (85) Google Scholar, 40Harada Y. Li H. Li H. Lennarz W.J. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.Proc. Natl. Acad. Sci. U S A. 2009; 106: 6945-6949Crossref PubMed Scopus (43) Google Scholar, 41Kreibich G. Freienstein C.M. Pereyra B.N. Ulrich B.L. Sabatini D.D. Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites.J. Cell Biol. 1978; 77: 488-506Crossref PubMed Scopus (83) Google Scholar, 42Levy R. Wiedmann M. Kreibich G. In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex.J. Biol. Chem. 2001; 276: 2340-2346Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 43Muller M. Blobel G. In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli.Proc. Natl. Acad. Sci. U S A. 1984; 81: 7421-7425Crossref PubMed Google Scholar, 44Savitz A.J. Meyer D.I. 180-kD ribosome receptor is essential for both ribosome binding and protein translocation.J. Cell Biol. 1993; 120: 853-863Crossref PubMed Scopus (0) Google Scholar, 45Tazawa S. Unuma M. Tondokoro N. Asano Y. Ohsumi T. Ichimura T. Sugano H. Identification of a membrane protein responsible for ribosome binding in rough microsomal membranes.J. Biochem. Tokyo. 1991; 109: 89-98PubMed Google Scholar). In this view, the SEC61 translocon serves a canonical role in secretory/membrane protein biogenesis by recruiting ribosomes engaged in the translation of this mRNA cohort, whereas other candidate ribosome interactors (e.g., p180, p34/LRRC59, SEC62) function as non-translocon ribosome binding sites. Ribosomes bound at these non-translocon sites may engage in the translation of both cytosolic and secretory/membrane protein-encoding transcripts. In the case of secretory/membrane polypeptides undergoing early elongation on non-translocon-associated ribosomes, signal sequence-bearing nascent chains might access translocons via lateral diffusion (21Jan C.H. Williams C.C. Weissman J.S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2014; 346: 1257521Crossref PubMed Scopus (168) Google Scholar, 25Reid D.W. Nicchitta C.V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2015; 16: 221-231Crossref PubMed Scopus (85) Google Scholar, 31Chartron J.W. Hunt K.C. Frydman J. Cotranslational signal-independent SRP preloading during membrane targeting.Nature. 2016; 536: 224-228Crossref PubMed Scopus (65) Google Scholar, 46Jan C.H. Williams C.C. Weissman J.S. LOCAL TRANSLATION. Response to comment on “Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2015; 348: 1217Crossref PubMed Scopus (0) Google Scholar, 47Reid D.W. Nicchitta C.V. LOCAL TRANSLATION. Comment on “Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2015; 348: 1217Crossref PubMed Scopus (0) Google Scholar). A primary prediction of this model is that different ribosome interacting proteins would reside in distinct membrane protein environments, perhaps reflecting the degree to which their bound ribosomes are dedicated to secretory/membrane protein synthesis. Using a BioID proximity-labeling approach, we recently reported that SEC61β, a translocon subunit, and the candidate ribosome-binding protein LRRC59 interact with populations of ribosomes engaged in the translation of divergent cohorts of mRNAs (34Hoffman A.M. Chen Q. Zheng T. Nicchitta C.V. Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis.J. Biol. Chem. 2019; 294: 8942-8958Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In this communication, we extend these studies by investigating the proximal ER protein interactomes of the four previously engineered BioID reporters (SEC61β, RPN1, SEC62, and LRRC59) (34Hoffman A.M. Chen Q. Zheng T. Nicchitta C.V. Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis.J. Biol. Chem. 2019; 294: 8942-8958Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In time course labeling studies, we observed that for each reporter, proximal interactome labeling intensified but only modestly diversified as a function of labeling time, a finding consistent with a functional domain organization of the ER. Unexpectedly, our data revealed that the previously reported ribosome receptor SEC62 interacts with functionally divergent protein networks, including those with roles in cell proliferation, signaling pathways, redox homeostasis, and cytoplasmic displaced ER luminal chaperones. In contrast, LRRC59 displays a highly SRP pathway-, translation-, and RNA-binding protein-enriched interactome. Both proximity proteomics and native immunoprecipitation studies found LRRC59 to interact almost exclusively with SRP machinery, non-canonical ER-RBPs, and translation initiation factors, suggesting a previously unappreciated role for LRRC59 in the organization and/or regulation of secretory/membrane protein synthesis on the ER. Consistent with this view, siRNA knockdown of LRRC59 expression substantially reduced protein synthesis levels in the cytosol and ER compartments. BirA-chimera constructs are described in (34Hoffman A.M. Chen Q. Zheng T. Nicchitta C.V. Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis.J. Biol. Chem. 2019; 294: 8942-8958Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). HEK293 Flp-InTM T-RExTM cell lines were generated according to the manufacturer's instructions (ThermoFisher Scientific). BirA-containing plasmids (0.4 µg), along with the pOGG4 (4 µg) plasmid were transfected into cells using 7.5 µL of Lipofectamine 3000 (ThermoFisher, L3000001). All transfections were performed in 6-well culture dishes at 80% confluency. Colonies were selected for between 48 hours and two weeks post-transfection using 100 μg/mL hygromycin (MediaTech, 30-240-CR, Manassas, VA) and 15 μg/mL blasticidin (ThermoFisher, R21001). A negative control cell line (“Empty Vector Control”) was generated by recombination of an empty vector pcDNA5-FRT/TO and antibiotic selection for an empty vector matched control. Cells were washed twice with ice-cold PBS containing 50 μg/mL of cycloheximide (CHX) (VWR, 94271, Radnor, PA) for 3 minutes. To extract the cytosolic (C) fraction, cells were permeabilized for 5–10 minutes at 4°C in buffer containing 110 mM KOAc, 25 mM HEPES pH 7.2, 2.5 mM MgCl2, 0.03% digitonin (Calbiochem, 3004010), 1 mM DTT, 50 μg/mL CHX, 40 U/mL RNAseOUT (Invitrogen, 10777-019, Carlsbad, CA), and protease inhibitor complex (PIC) (Sigma Aldrich, P8340). Supernatants were collected as the cytosolic fraction, and cells were then rinsed with wash buffer (110 mM KOAc, 25 mM HEPES pH 7.2, 2.5 mM Mg(OAc)2, 0.004% to 0.008% digitonin, 1 mM DTT, 50 μg/mL CHX, 40 U/mL RNAseOUT, and PIC). To extract the membrane (M) fraction, the washed cells were then lysed either for 5 minutes at 4°C in lysis buffer 1 (400 mM KOAc, 25 mM HEPES pH 7.2, 15 mM MgCl2, 1% NP-40, 0.5% DOC, 1 mM DTT, 50 μg/mL CHX, 40 U/mL RNAseOUT, and PIC) or for 15 minutes at 4°C in lysis buffer 2 (200 mM KCl, 25 mM HEPES pH 7.2, 15 mM MgCl2, 1 mM DTT, 2% dodecylmaltoside (DDM), 50 µg/mL CHX, 40 U/ml RNaseOUT, and 1X protease inhibitor). Subcellular fractions were cleared by centrifugation (15,300 × g for 10 minutes). Total cell lysis was performed by incubating cells at 4°C for 10 minutes in membrane lysis buffer 1, followed by centrifugation at 15,300 × g for 10 minutes. Canine pancreas rough microsomes (RM) (48Walter P. Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.Proc. Natl. Acad. Sci. U S A. 1980; 77: 7112-7116Crossref PubMed Google Scholar) were adjusted to a concentration of 4 mg/mL in 500 μL of BirA reaction buffer (20 mM Tris pH 8, 5 mM CaCl2, 100 mM KCl2, 10 mM MgCl2, 3 mM ATP, 1.5 mM biotin, 5 mM phosphocreatine (Sigma-Aldrich, P7936), and 5 μg/mL of creatine kinase (Sigma-Aldrich, C3755)). Purified recombinant BirA*-GST fusion protein was added at a concentration of 10 μg/mL. Following 0, 1, 3, 6, and 18 hours, 100 μL of reaction was removed, flash frozen in a dry ice/ethanol bath, and stored at –80°C for subsequent analysis. Protein lysate concentrations were determined using a Pierce BCA Protein Assay Kit (ThermoFisher, 23225). Proteins were resolved by SDS-PAGE using either 10% acrylamide gels or 12% acrylamide gels containing 0.5% trichloroethanol. Gels were UV irradiated for 5 minutes and imaged using an Amersham Imager 600 (GE Life Sciences). Gels or membranes were then equilibrated in Tris-glycine transfer buffer for 5-10 minutes and transferred onto nitrocellulose membranes. Membranes were blocked in 3% BSA and probed for BirA (Abcam, ab14002), streptavidin-RD680 (Li-Cor, P/N 925-68079; 1:20,000), TRAPα (49Migliaccio G. Nicchitta C.V. Blobel G. The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation.J. Cell Biol. 1992; 117: 15-25Crossref PubMed Google Scholar), GRP94 (50Jagannathan S. Nwosu C. Nicchitta C.V. Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.Methods" @default.
• W3048449614 created "2020-08-18" @default.
• W3048449614 creator A5030163108 @default.
• W3048449614 creator A5032530883 @default.
• W3048449614 creator A5036096197 @default.
• W3048449614 creator A5059378741 @default.
• W3048449614 creator A5059684381 @default.
• W3048449614 date "2020-11-01" @default.
• W3048449614 modified "2023-09-28" @default.
• W3048449614 title "Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane" @default.
• W3048449614 cites W10583676 @default.
• W3048449614 cites W156511211 @default.
• W3048449614 cites W1571379445 @default.
• W3048449614 cites W1583738858 @default.
• W3048449614 cites W1586371368 @default.
• W3048449614 cites W1637568577 @default.
• W3048449614 cites W1666668357 @default.
• W3048449614 cites W1969412365 @default.
• W3048449614 cites W1973463353 @default.
• W3048449614 cites W1974130356 @default.
• W3048449614 cites W1974378947 @default.
• W3048449614 cites W1975567952 @default.
• W3048449614 cites W1979260907 @default.
• W3048449614 cites W1981610784 @default.
• W3048449614 cites W1989616670 @default.
• W3048449614 cites W1991655565 @default.
• W3048449614 cites W1994434920 @default.
• W3048449614 cites W2010005484 @default.
• W3048449614 cites W2011112307 @default.
• W3048449614 cites W2012034410 @default.
• W3048449614 cites W2013219100 @default.
• W3048449614 cites W2015556811 @default.
• W3048449614 cites W2023261130 @default.
• W3048449614 cites W2023299080 @default.
• W3048449614 cites W2032362466 @default.
• W3048449614 cites W2034899234 @default.
• W3048449614 cites W2035671523 @default.
• W3048449614 cites W2037288722 @default.
• W3048449614 cites W2037870039 @default.
• W3048449614 cites W2038254381 @default.
• W3048449614 cites W2038505563 @default.
• W3048449614 cites W2039812608 @default.
• W3048449614 cites W2040119910 @default.
• W3048449614 cites W2049231436 @default.
• W3048449614 cites W2051250838 @default.
• W3048449614 cites W2052826009 @default.
• W3048449614 cites W2063258048 @default.
• W3048449614 cites W2064254630 @default.
• W3048449614 cites W2068417373 @default.
• W3048449614 cites W2068663593 @default.
• W3048449614 cites W2071030970 @default.
• W3048449614 cites W2078824282 @default.
• W3048449614 cites W2083197833 @default.
• W3048449614 cites W2083323375 @default.
• W3048449614 cites W2086207586 @default.
• W3048449614 cites W2091549056 @default.
• W3048449614 cites W2095701936 @default.
• W3048449614 cites W2096321546 @default.
• W3048449614 cites W2099923841 @default.
• W3048449614 cites W2099951367 @default.
• W3048449614 cites W2100300856 @default.
• W3048449614 cites W2101060338 @default.
• W3048449614 cites W2105707485 @default.
• W3048449614 cites W2105729094 @default.
• W3048449614 cites W2106969933 @default.
• W3048449614 cites W2109184486 @default.
• W3048449614 cites W2113942795 @default.
• W3048449614 cites W2117761303 @default.
• W3048449614 cites W2117986250 @default.
• W3048449614 cites W2120758664 @default.
• W3048449614 cites W2122550753 @default.
• W3048449614 cites W2122670476 @default.
• W3048449614 cites W2123658589 @default.
• W3048449614 cites W2124821829 @default.
• W3048449614 cites W2127387423 @default.
• W3048449614 cites W2128207929 @default.
• W3048449614 cites W2133469761 @default.
• W3048449614 cites W2134091981 @default.
• W3048449614 cites W2134257152 @default.
• W3048449614 cites W2138630554 @default.
• W3048449614 cites W2139070887 @default.
• W3048449614 cites W2141300290 @default.
• W3048449614 cites W2153349010 @default.
• W3048449614 cites W2153545502 @default.
• W3048449614 cites W2156133814 @default.
• W3048449614 cites W2160340248 @default.
• W3048449614 cites W2164778558 @default.
• W3048449614 cites W2167844024 @default.
• W3048449614 cites W2171030481 @default.
• W3048449614 cites W2181567397 @default.
• W3048449614 cites W2259709654 @default.
• W3048449614 cites W2261446217 @default.
• W3048449614 cites W2288568691 @default.
• W3048449614 cites W2298917269 @default.
• W3048449614 cites W2416403255 @default.
• W3048449614 cites W2461183258 @default.
• W3048449614 cites W2485148075 @default.
• W3048449614 cites W2502120512 @default.

• next