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 b5, carrying an N-glycosylation site in its C-terminal domain (b5-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 b5-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 b5-Nglyc translocation. The kinetics of translocation were faster for b5-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 b5-Nglyc. Similarly, only low ATP concentrations (below 1 μM), in addition to cytosolic protein(s), were required for in vitro translocation of b5-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored b5-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.
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