Translocation of the C terminus of a tail-anchored protein across the endoplasmic reticulum membrane in yeast mutants defective in signal peptide-driven translocation

被引:54
作者
Yabal, M
Brambillasca, S
Soffientini, P
Pedrazzini, E
Borgese, N
Makarow, M
机构
[1] CNR, Inst Neurosci, Cellular & Mol Pharmacol Sect, I-20129 Milan, Italy
[2] Univ Catanzaro Magna Graecia, Fac Pharm, I-8021 Roccelletta di Borgia, Catanzaro, Italy
关键词
D O I
10.1074/jbc.M210253200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus. Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s). Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc). Utilization of this site was adopted as a rigorous criterion for translocation across the ER membrane of yeast wild-type and mutant cells. The C terminus of b(5)-Nglyc was rapidly glycosylated in mutants where Sec61p was defective and incapable of translocating carboxypeptidase Y, a well known substrate for post-translational translocation. Likewise, inactivation of several other components of the translocon machinery had no effect on b(5)-Nglyc translocation. The kinetics of translocation were faster for b(5)-Nglyc than for a signal peptide-containing reporter. Depletion of the cellular ATP pool to a level that retarded Sec61p-dependent post-translational translocation still allowed translocation of b(5)-Nglyc. Similarly, only low ATP concentrations (below I pm), in addition to cytosolic protein(s), were required for in vitro translocation of b(5)-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored b(5)-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.
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页码:3489 / 3496
页数:8
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