Abstract
During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro(178) and Pro(183) of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.
Highlights
selenoprotein S (SelS) is a binding partner of p97(VCP) for endoplasmic reticulum-associated degradation (ERAD)
We found that the expression levels of SelS, glucose-regulated protein 78 (GRP78, an ER chaperone), C/EBPhomologous protein (CHOP, a pro-apoptosis protein), cleaved caspase12 (c-casp12, an ER stress-dependent pro-apoptosis protein), and full-length and cleaved caspase3 (f- and c-casp3, respectively, critical executioners of apoptosis) increased in N2a cells when treated with 1 g/ml Tm or 100 nM Tg for 24 h
Localization of p97(VCP) in the membrane did not increase in SelS knockdown cells (Fig. 1F); the cytosolic p97(VCP) level was higher in SelS knockdown cells than that in control cells under ER stress conditions
Summary
SelS is a binding partner of p97(VCP) for endoplasmic reticulum-associated degradation (ERAD). During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd, and gp. The proline mutants did not rescue ER stress in SelS knockdown cells These results suggest that both Pro178 and Pro183 of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress. SelS, which is known as VIMP (VCP-interacting membrane protein), is a selenoprotein that contains a single selenocysteine (Sec, U) at position 188 as a C-terminal penultimate residue (7–9). To understand the regulatory function of SelS in ERAD, we identified the effect of the protein on ER stress and the essential residues of SelS for binding to p97(VCP) in this study
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