Sec62 as a novel gating modulator for protein translocation and ion permeability of the mammalian Sec61 complex (LB196)

Abstract

In mammalian cells the endoplasmic reticulum (ER) plays a key role during the biogenesis of secretory and membrane proteins and acts as a major regulator for intracellular Ca2+ signaling. Sec62, a component of the ER‐protein translocase, acts during post‐translational translocation of small precursor proteins and in ER‐Ca2+ leakage‐control. Our project aims to elucidate the molecular mechanisms how it regulates the translocation machinery concerning these two fundamental and seemingly opposing functions. We used siRNA mediated depletion of target proteins from human cells followed by functional assays for protein transport and live cell calcium imaging.Depletion of Sec62 in combination with proteasomal inhibition led to accumulation of the precursor of the ER resident protein ERj3 at the cytosolic face of the ER membrane. Control experiments using different siRNA as well as a plasmid rescue approach confirmed the specificity for Sec62. Follow‐up experiments revealed a pattern of dependencies upon translocon components which is characteristic for a post‐translational translocation pathway.Decrease of Sec62 level led to an increased ER calcium leakage that could be attributed to Sec61α. We observed that Sec62 binds the N‐terminus of Sec61α in a Ca2+ sensitive manner and identified a crucial putative EF‐hand in the C‐terminal part of Sec62.These experiments demonstrated for the first time that human Sec62 is important for the early steps of translocation of a large precursor protein indicating that Sec62 participation is not restricted to small proteins as suggested in previous studies.Additionally, Sec62 acts as a modulator, shifting the translocon from the open to the closed state after translocation is completed in order to maintain the ion impermeability of the ER membrane.Further experiments will allow us to determine the recently proposed role of Sec62 for various cancer entities.