Biochemical characterization of the vacuolar palmitoyl acyltransferase.
Vacuole fusion requires Sec18p-dependent acylation of the armadillo-repeat protein Vac8p that has been isolated with cis-SNARE complexes. To gain more insight into the mechanism of acylation, we analyzed the palmitoylation reaction on isolated vacuoles or in vacuolar detergent extracts. Recombinant Vac8p is palmitoylated when added to vacuoles and is anchored to membranes after modification. The palmitoyl acyltransferase (PAT) extracted from vacuolar membranes is functional in detergent extracts and shows all characteristics of an enzymatic activity: It modifies exogenous Vac8p in a temperature-, dose- and time-dependent manner, and is sensitive to bromo-palmitate, a known inhibitor of protein palmitoylation in vivo. Importantly, PAT is specific for palmitoyl-CoA, since myristoyl- and stearyl-CoA can compete with labeled Pal-CoA only at 10-fold higher amounts.
Veit
Michael
M
Department of Immunology and Molecular Biology, Vet-Med Faculty of the Free University Berlin, Philippstrasse 13, 10115 Berlin, Germany.
Dietrich
Lars E P
LE
Ungermann
Christian
C
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
D011994:Recombinant Proteins; D000217:Acyltransferases