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1.
PLoS One ; 8(10): e75394, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24130708

RESUMO

The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Transporte Proteico/fisiologia , RNA Interferente Pequeno
2.
J Mol Biol ; 423(4): 590-9, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22846909

RESUMO

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady-state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Neurospora crassa/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Mol Biol Cell ; 21(5): 691-703, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20071467

RESUMO

Because of similarity to their yeast orthologues, the two membrane proteins of the human endoplasmic reticulum (ER) Sec62 and Sec63 are expected to play a role in protein biogenesis in the ER. We characterized interactions between these two proteins as well as the putative interaction of Sec62 with ribosomes. These data provide further evidence for evolutionary conservation of Sec62/Sec63 interaction. In addition, they indicate that in the course of evolution Sec62 of vertebrates has gained an additional function, the ability to interact with the ribosomal tunnel exit and, therefore, to support cotranslational mechanisms such as protein transport into the ER. This view is supported by the observation that Sec62 is associated with ribosomes in human cells. Thus, the human Sec62/Sec63 complex and the human ER membrane protein ERj1 are similar in providing binding sites for BiP in the ER-lumen and binding sites for ribosomes in the cytosol. We propose that these two systems provide similar chaperone functions with respect to different precursor proteins.


Assuntos
Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Drosophila melanogaster , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , Humanos , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície
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