Translocation
of the C-terminus of a tail-anchored protein across the ER membrane in yeast
mutants defective in signal peptide-driven translocation.
Monica Yabal (1),
Nica Borgese (2), Marja Makarow (1)
(1) Prog.of Cellular Biotechnology, Institute of Biotechnology, Viikinkaari 9,
Helsinki, 00014, Finland (monica.yabal@helsinki.fi); (2) Consiglio Nazionale
delle Ricerche Institute of Neuroscience, Cellular and Molecular Pharmacology
Section, Via Vanvitelli 32 - 20129 Milano, Italy
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 the lipid bilayer by poorly understood
post-translational mechanism(s). Translocation of the C-terminus of the
mammalian ER isoform of cytochrome b(5), carrying an N-glycosylation site in
its C-terminal domain (b(5)-Nglyc) to monitor translocation across the ER
membrane, was studied in living S. cerevisiae cells. The C-terminus of
b(5)-Nglyc was rapidly translocated in mutants where the Sec61 translocon was
defective, resulting in cytosolic accumulation of carboxypeptidase Y (CPY).
Likewise, inactivation of several other components of the post-translocational
translocon machinery (Sec62p/63p and Kar2p) had no effect on b(5)-Nglyc
translocation. Translocation kinetics of b(5)-Nglyc were faster than those of a
signal peptide-containing reporter. Depletion of the cellular ATP pool to a
level which retarded Sec61p-dependent post-translational translocation still
allowed translocation of b(5)-Nglyc. Thus, translocation of c-tail-anchored
b(5)-Nglyc proceeds by a mechanism different from that utilized in signal
peptide-driven post-translational translocation, in living cells.