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Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells.
Proc Natl Acad Sci U S A. 2020 Jul 14; 117(28):16401-16408.PN

Abstract

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.

Authors+Show Affiliations

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi 980-8577, Japan hiroshi.kadokura.b3@tohoku.ac.jp.Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi 980-8577, Japan.Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi 980-8577, Japan.Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi 980-8577, Japan.Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi 980-8577, Japan.Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi 980-8577, Japan.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32601215

Citation

Kadokura, Hiroshi, et al. "Observing the Nonvectorial yet Cotranslational Folding of a Multidomain Protein, LDL Receptor, in the ER of Mammalian Cells." Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 28, 2020, pp. 16401-16408.
Kadokura H, Dazai Y, Fukuda Y, et al. Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells. Proc Natl Acad Sci USA. 2020;117(28):16401-16408.
Kadokura, H., Dazai, Y., Fukuda, Y., Hirai, N., Nakamura, O., & Inaba, K. (2020). Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells. Proceedings of the National Academy of Sciences of the United States of America, 117(28), 16401-16408. https://doi.org/10.1073/pnas.2004606117
Kadokura H, et al. Observing the Nonvectorial yet Cotranslational Folding of a Multidomain Protein, LDL Receptor, in the ER of Mammalian Cells. Proc Natl Acad Sci USA. 2020 Jul 14;117(28):16401-16408. PubMed PMID: 32601215.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells. AU - Kadokura,Hiroshi, AU - Dazai,Yui, AU - Fukuda,Yo, AU - Hirai,Naoya, AU - Nakamura,Orie, AU - Inaba,Kenji, Y1 - 2020/06/29/ PY - 2020/12/29/pmc-release PY - 2020/7/1/pubmed PY - 2020/7/1/medline PY - 2020/7/1/entrez KW - LDL receptor KW - cotranslational folding KW - disulfide bonds KW - multidomain protein KW - nonvectorial folding SP - 16401 EP - 16408 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 28 N2 - Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells. SN - 1091-6490 UR - https://www.unboundmedicine.com/medline/citation/32601215/Observing_the_nonvectorial_yet_cotranslational_folding_of_a_multidomain_protein,_LDL_receptor,_in_the_ER_of_mammalian_cells L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=32601215 DB - PRIME DP - Unbound Medicine ER -
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