Feldheim D, Rothblatt J, Schekman R
Howard Hughes Medical Research Institute, Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Mol Cell Biol. 1992 Jul;12(7):3288-96. doi: 10.1128/mcb.12.7.3288-3296.1992.
SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.
SEC63编码一种将分泌蛋白转运到酿酒酵母内质网（ER）中所需的蛋白质（J. A. Rothblatt、R. J. Deshaies、S. L. Sanders、G. Daum和R. Schekman，《细胞生物学杂志》109:2641 - 2652，1989年）。针对该蛋白重组形式的抗体可检测到一种73 kDa的多肽，通过免疫荧光显微镜观察，该多肽定位于核膜 - 内质网网络。细胞分级分离和蛋白酶保护实验证实了Sec63p是一种整合膜蛋白的预测。构建了一系列SEC63 - SUC2融合基因以评估Sec63p在内质网膜中的拓扑结构。最大的杂合蛋白未被糖基化，这表明Sec63p的羧基末端面向胞质溶胶。将蔗糖酶与Sec63p中由两个假定跨膜结构域侧翼的环融合产生一种高度糖基化的杂合蛋白。这个环与大肠杆菌热休克蛋白DnaJ的氨基末端同源，可能面向内质网腔。类似于大肠杆菌中DnaJ和Hsp70样DnaK蛋白的相互作用，Sec63p的DnaJ环可能将腔内Hsp70（BiP/GRP78/Kar2p）募集到转运装置中。DnaJ环的两个高度保守位置的突变和羧基末端的短缺失会使Sec63p活性失活。Sec63p与其他几种蛋白质相关联，包括31.5 kDa的糖蛋白Sec61p和一种23 kDa的蛋白质，并且与这些蛋白质一起可能构成多肽转运装置的一部分。一个无功能的DnaJ结构域突变等位基因不会干扰Sec63p/Sec61p/gp31.5/p23复合物的形成。
