Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane
| Autor: | Molly M. Hannigan, J. Will Thompson, Christopher V. Nicchitta, Tianli Zheng, Alyson M. Hoffman |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Proteomics
Proteasome Endopeptidase Complex Proteome Endoplasmic Reticulum Biochemistry Interactome Mass Spectrometry Analytical Chemistry 03 medical and health sciences Cytosol SEC62 Cell Line Tumor Protein biosynthesis Humans Gene Silencing Protein Interaction Maps RNA Small Interfering Molecular Biology Integral membrane protein 030304 developmental biology 0303 health sciences biology Chemistry Research Endoplasmic reticulum Cytoplasmic translation 030302 biochemistry & molecular biology Oligosaccharyltransferase Computational Biology Membrane Proteins Membrane Transport Proteins Translation (biology) Recombinant Proteins Cell biology Gene Ontology Membrane protein Chaperone (protein) Protein Biosynthesis biology.protein Oxidation-Reduction Ribosomes SEC Translocation Channels |
| Zdroj: | Mol Cell Proteomics |
| ISSN: | 1535-9476 |
| DOI: | 10.1074/mcp.ra120.002228 |
| Popis: | 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, [(35)S]-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. |
| Databáze: | OpenAIRE |
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