Vac8p function: analysis of the roles of palmitoylation and the
armadillo repeats.
Emily Kauffman, Yutian Peng, Yong-Xu Wang, Fusheng Tang, Lois
Weisman
Department of Biochemistry, University of Iowa, 51 Newton Road, Iowa
City, IA 52242, USA
Vac8p, a vacuole membrane protein in Saccharomyces cerevisiae, is
required for vacuole inheritance, cytoplasm to vacuole protein targeting
(Cvt) and nuclear-vacuole junction formation. Vac8p is myristoylated and
palmitoylated at its amino-terminus and these modifications are required
both for Vac8p localization on the vacuole membrane and for all its
functions except for the Cvt pathway. Three cysteines at the N-terminus
are reversibly palmitoylated. To investigate how many palmitoyl moieties
are required for Vac8p function, we generated all possible single and
double point mutations to create Vac8p mutants with one or two palmitoyl
groups. The effect on Vac8p function directly correlates with the number
of palmitoyl moieties. The position of the palmitates is also important.
The rest of the Vac8p sequence is comprised of eleven armadillo repeats
(ARM), which function as a docking site for multiple binding partners,
each of which functions in a distinct Vac8p mediated process. Deletion
of ARM2 abolishes all Vac8p functions tested. In contrast, deletion of
ARM9-11 has no affect. We recently discovered that Vac8p is also
required for homotypic vacuole fusion. Vac8p is not necessary for
priming and docking but acts after SNARE pairing. We hypothesize that
through its armadillo repeats Vac8p binds to a yet unidentified
component of the fusion machinery and through its palmitoyl moieties
brings the machinery to a specific fusogenic lipid domain.
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