The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8.
Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3/Ynl326c is required for efficient localization of Vac8 to vacuoles in vivo, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuole-associated Vac8 appears to be palmitoylated in a pfa3 mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate/palmitate (as in Vac8) but also one that is targeted by a myristate/basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole.
Hou
Haitong
H
Biochemie-Zentrum der Universität Heidelberg, Germany.
Subramanian
Kanagaraj
K
LaGrassa
Tracy J
TJ
Markgraf
Daniel
D
Dietrich
Lars E P
LE
Urban
Jörg
J
Decker
Nadine
N
Ungermann
Christian
C
D000217:Acyltransferases / Q000378:metabolism*; D019921:Biotinylation; D047389:Casein Kinase I / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D008074:Lipoproteins; C501979:MEH1 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C111373:VAC8 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D010171:Palmitoyl Coenzyme A; D000217:Acyltransferases; C505807:Pfa3 protein, S cerevisiae; D047389:Casein Kinase I; C479667:YCK3 protein, S cerevisiae