Lee Jea Hwang, Kwon Joon Hyun, Jeon Yeong Ha, Ko Kwan Young, Lee Seung-Rock, Kim Ick Young
From the Laboratory of Cellular and Molecular Biochemistry, Division of Life Sciences, Korea University, 1, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea and.
the Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Science, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
J Biol Chem. 2014 May 16;289(20):13758-68. doi: 10.1074/jbc.M113.534529. Epub 2014 Apr 3.
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.
在内质网（ER）相关降解过程中，p97（VCP）通过与跨膜蛋白相互作用被招募到内质网膜上，这些跨膜蛋白包括硒蛋白S（SelS）、硒蛋白K（SelK）、hrd1和gp78。SelS具有一个单次跨膜结构域，并通过与p97（VCP）相互作用保护细胞免受内质网应激诱导的凋亡。SelS的胞质尾由一个卷曲螺旋结构域、一个假定的VCP相互作用基序（VIM）和一个不明显的富含甘氨酸和脯氨酸的二级结构组成。为了了解内质网应激期间SelS的调控机制，我们使用小鼠神经母细胞瘤细胞和人胚肾293细胞研究了该蛋白与p97（VCP）的相互作用。诱导内质网应激时，SelS的表达水平升高。此外，在内质网应激的作用增强时，SelS敲低细胞中p97（VCP）向内质网膜的募集受到抑制。通过异位表达SelS U188C可挽救SelS敲低的影响。p97（VCP）与SelS U188C相互作用并被招募到内质网膜。SelS的VIM缺失突变体SelS[ΔVIM]的表达在细胞中也显示出挽救作用和与p97（VCP）的相互作用。然而，将SelS的脯氨酸残基位置178或183突变为丙氨酸或缺失的突变体不与p97（VCP）相互作用。脯氨酸突变体不能挽救SelS敲低细胞中的内质网应激。这些结果表明，SelS的Pro（178）和Pro（183）在p97（VCP）向内质网膜的转运中起重要作用，并保护细胞免受内质网应激。
