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Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP r...

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Autores principales: Yi, Jae Kyo, Fujita, Hidenobu, Mandon, Elisabet C., Gilmore, Reid
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503631/
https://www.ncbi.nlm.nih.gov/pubmed/34492269
http://dx.doi.org/10.1016/j.jbc.2021.101171
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author Yi, Jae Kyo
Fujita, Hidenobu
Mandon, Elisabet C.
Gilmore, Reid
author_facet Yi, Jae Kyo
Fujita, Hidenobu
Mandon, Elisabet C.
Gilmore, Reid
author_sort Yi, Jae Kyo
collection PubMed
description The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.
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spelling pubmed-85036312021-10-18 Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins Yi, Jae Kyo Fujita, Hidenobu Mandon, Elisabet C. Gilmore, Reid J Biol Chem Research Article The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences. American Society for Biochemistry and Molecular Biology 2021-09-04 /pmc/articles/PMC8503631/ /pubmed/34492269 http://dx.doi.org/10.1016/j.jbc.2021.101171 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Yi, Jae Kyo
Fujita, Hidenobu
Mandon, Elisabet C.
Gilmore, Reid
Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins
title Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins
title_full Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins
title_fullStr Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins
title_full_unstemmed Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins
title_short Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins
title_sort rapid inactivation of the yeast sec complex selectively blocks transport of post-translationally translocated proteins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503631/
https://www.ncbi.nlm.nih.gov/pubmed/34492269
http://dx.doi.org/10.1016/j.jbc.2021.101171
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