Translocation of the C Terminus of a Tail-anchored Protein across the Endoplasmic Reticulum Membrane in Yeast Mutants Defective in Signal Peptide-driven Translocation

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 1 μ m ), 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.