Palmitoylation determines the function of Vac8 at the yeast vacuole.
Palmitoylation stably anchors specific proteins to membranes, but may also have a direct effect on the function of a protein. The yeast protein Vac8 is required for efficient vacuole fusion, inheritance and cytosol-to-vacuole trafficking. It is anchored to vacuoles by an N-terminal myristoylation site and three palmitoylation sites, also known as the SH4 domain. Here, we address the role of Vac8 palmitoylation and show that the position and number of substrate cysteines within the SH4 domain determine the vacuole localization of Vac8: stable vacuole binding of Vac8 requires two cysteines within the N-terminus, regardless of the combination. Importantly, our data suggest that palmitoylation adds functionality to Vac8 beyond simple localization. A mutant Vac8 protein, in which the palmitoylation sites were replaced by a stretch of basic residues, still localizes to vacuole membranes and functions in cytosol-to-vacuole transport, but can only complement the function of Vac8 in morphology and inheritance if it also contains a single cysteine within the SH4 domain. Our data suggest that palmitoylation is not a mere hydrophobic anchor required solely for localization, but influences the protein function(s).
Subramanian
Kanagaraj
K
Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Dietrich
Lars E P
LE
Hou
Haitong
H
LaGrassa
Tracy J
TJ
Meiringer
Christoph T A
CT
Ungermann
Christian
C
D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000502:physiology*; D033921:Vesicular Transport Proteins; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology
D008074:Lipoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C111373:VAC8 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D019308:Palmitic Acid; D003545:Cysteine