Role of human sec63 in modulating the steady-state levels of multi-spanning membrane proteins

PLoS One. 2012;7(11):e49243. doi: 10.1371/journal.pone.0049243. Epub 2012 Nov 15.

Abstract

The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning ER reporters are not affected. Consistent with this, the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levels independent of its Sec62-interacting motif, indicating that it may not act in conjunction with Sec62 in human cells. The specific action of Sec63 is further sustained by our observations that the up-regulation of either Sec62 or two other ER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. A J domain-specific mutation of Sec63, proposed to weaken its interaction with the ER resident BiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these results suggest that Sec63 may perform a substrate-selective quantity control function during cotranslational ER import.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins
  • Blotting, Western
  • Cell Line
  • DNA Primers / genetics
  • Endoplasmic Reticulum / metabolism*
  • Gene Expression Regulation / genetics
  • Gene Expression Regulation / physiology*
  • Heat-Shock Proteins / metabolism
  • Humans
  • Luminescent Proteins
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins / metabolism
  • Microscopy, Fluorescence
  • Molecular Chaperones
  • Mutation / genetics
  • RNA, Small Interfering / genetics
  • RNA-Binding Proteins
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transfection
  • Viral Envelope Proteins / metabolism

Substances

  • Bacterial Proteins
  • DNA Primers
  • Heat-Shock Proteins
  • Luminescent Proteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Molecular Chaperones
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • S envelope protein, hepatitis B virus
  • SEC62 protein, human
  • SEC63 protein, human
  • Viral Envelope Proteins
  • yellow fluorescent protein, Bacteria
  • molecular chaperone GRP78

Grant support

This work was supported in part by grants to RP from the Deutsche Forschungsgemeinschaft (PR305/1-3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.