Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.
Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.
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DOI：
10.1083/jcb.120.1.95
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发表时间：
1993-01
期刊：
影响因子：
--
通讯作者：
Schekman R
中科院分区：
文献类型：
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作者：
Brodsky JL;Hamamoto S;Feldheim D;Schekman R
我们使用与大豆磷脂形成囊泡的酵母微粒体去污剂可溶性部分重建了前α因子原的移位和信号肽加工。重建的易位需要ATP，并且当sec63和kar2（BiP）突变体细胞用作膜的来源时是缺乏的。观察到正常的易位与囊泡重建从纯野生型酵母BiP和可溶性级分的kar2突变膜的混合物。另外两个热休克同源物（hsc）70同源物，酵母胞质hsc 70（Ssalp）和E. coli dnaK蛋白不能替代BiP。相反，BiP是没有活性的条件下，易位到天然ER囊泡需要胞质hsc70。我们的结论是，胞浆hsc70和BiP服务不可互换的作用，在多肽易位，可能是因为不同的，不对称取向的膜蛋白需要招募每个蛋白质的ER膜的相对表面。
We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.