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Molecular and Cellular Biology, July 2000, p. 5350-5359, Vol. 20, No. 14
Department of
Pharmacology1 and Departments of Surgery
and Pharmacology,2 Wayne State University
School of Medicine, Detroit, Michigan 48201
Received 22 November 1999/Returned for modification 30 December
1999/Accepted 17 April 2000
Ubiquitination of the plasma membrane-localized yeast a-factor
receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation. This report identifies two mutants
that block uptake by blocking ubiquitination, these being mutant either
for the ankyrin repeat protein Akr1p or for the redundant type I casein
kinases Yck1p and Yck2p. While no obvious defect was seen for wild-type
Ste3p phosphorylation in akr1 or yck mutant
backgrounds, examination of the
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Akr1p and the Type I Casein Kinases Act prior to the
Ubiquitination Step of Yeast Endocytosis: Akr1p Is Required for
Kinase Localization to the Plasma Membrane
320-413 Ste3p deletion mutant
phosphorylation did reveal a clear defect in both mutants. The
320-413 deletion removes 18 Ser-Thr residues (possible
YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr
residues of the Ste3p PEST-like ubiquitination-endocytosis signal. Two
other phenotypes link akr1 and yck mutants:
both are defective in phosphorylation of wild-type
-factor receptor,
and while both are defective for Ste3p constitutive internalization,
both remain partially competent for the Ste3p ligand-dependent uptake
mode. Yck1p-Yck2p may be the function responsible in phosphorylation of
the PEST-like ubiquitination-endocytosis signal. Akr1p appears to
function in localizing Yck1p-Yck2p to the plasma membrane, a
localization that depends on prenylation of C-terminal dicysteinyl
motifs. In akr1
cells, Yck2p is mislocalized, showing a
diffuse cytoplasmic localization identical to that seen for a Yck2p
mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p
requirement for the lipid modification of Yck2p, for prenylation, or
possibly for palmitoylation.
*
Corresponding author. Mailing address: Departments of
Surgery and Pharmacology, Wayne State University School of Medicine, Elliman Building, Room 1205, 421 E. Canfield, Detroit, MI 48201. Phone:
(313) 577-7807. Fax: (313) 577-7642. E-mail:
ndavis{at}cmb.biosci.wayne.edu.
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