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J. Biol. Chem., Vol. 278, Issue 5, 3489-3496, January 31, 2003
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§,
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, and
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From the 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.
Program of Cellular Biotechnology, Institute
of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland, ¶ Consiglio Nazionale delle Ricerche Institute
of Neuroscience, Cellular and Molecular Pharmacology Section, Via
Vanvitelli 32-20129 Milano, Italy, and the ** Faculty of
Pharmacy, University of Catanzaro "Magna Graecia," 8021 Roccelletta di Borgia (Catanzaro), Italy
Present address: Consiglio Nazionale delle Ricerche Istituto
di Biologia e Biotecnologia Agraria, Via Bassini 15-20133 Milano, Italy.

To whom correspondence should be addressed. Tel.:
39-02-50316971; Fax: 39-02-7490574; E-mail: nica@csfic.mi.cnr.it.
§§
A Biocentrum Helsinki fellow.
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