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1.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38866426

RESUMO

Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Ribossomos , Ribossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Microscopia Crioeletrônica , Canais de Translocação SEC/metabolismo , Canais de Translocação SEC/química , Chaperonas Moleculares/metabolismo , Transporte Proteico , Modelos Moleculares
2.
Nature ; 614(7946): 160-167, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36697828

RESUMO

The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3-7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.


Assuntos
Retículo Endoplasmático , Membranas Intracelulares , Biossíntese de Proteínas , Humanos , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Membranas Intracelulares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Guanosina Trifosfato/metabolismo , Complexos Multiproteicos/metabolismo
3.
J Cell Sci ; 133(3)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019826

RESUMO

The endoplasmic reticulum (ER) translocon complex is the main gate into the secretory pathway, facilitating the translocation of nascent peptides into the ER lumen or their integration into the lipid membrane. Protein biogenesis in the ER involves additional processes, many of them occurring co-translationally while the nascent protein resides at the translocon complex, including recruitment of ER-targeted ribosome-nascent-chain complexes, glycosylation, signal peptide cleavage, membrane protein topogenesis and folding. To perform such varied functions on a broad range of substrates, the ER translocon complex has different accessory components that associate with it either stably or transiently. Here, we review recent structural and functional insights into this dynamically constituted central hub in the ER and its components. Recent cryo-electron microscopy (EM) studies have dissected the molecular organization of the co-translational ER translocon complex, comprising the Sec61 protein-conducting channel, the translocon-associated protein complex and the oligosaccharyl transferase complex. Complemented by structural characterization of the post-translational import machinery, key molecular principles emerge that distinguish co- and post-translational protein import and biogenesis. Further cryo-EM structures promise to expand our mechanistic understanding of the various biochemical functions involving protein biogenesis and quality control in the ER.


Assuntos
Retículo Endoplasmático , Microscopia Crioeletrônica , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático Rugoso/metabolismo , Transporte Proteico , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo
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