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 microm), 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.
Yabal
Monica
M
Program of Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland.
Brambillasca
Silvia
S
Soffientini
Paolo
P
Pedrazzini
Emanuela
E
Borgese
Nica
N
Makarow
Marja
M
D000595:Amino Acid Sequence; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017353:Gene Deletion; D005838:Genotype; D006031:Glycosylation; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015786:Cytochromes b5