Abstract
The formation of dimers or higher-order oligomers is a property of numerous integral membrane proteins, including ion channels, transporters, and receptors. In this study, we examined whether members of the DHHC-S-acyltransferase family oligomerize in intact cells and in vitro. DHHC-S-acyltransferases are integral membrane proteins that catalyze the addition of palmitate to cysteine residues on proteins at the cytoplasmic face of cell membranes. Bioluminescence resonance energy transfer (BRET) experiments revealed that DHHC2 or DHHC3 (Golgi-specific DHHC zinc finger protein (GODZ)) self-associate when expressed in HEK-293 cells. Homomultimer formation was confirmed by coimmunoprecipitation. Purified DHHC3 resolved predominately as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild-type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of its lipid substrate palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Enzyme activity of a covalently linked DHHC3 dimer was less than that of the monomeric form, suggesting that enzyme activity may be modulated by the oligomerization status of the protein.
Highlights
Oligomerization of DHHC palmitoyltransferases has been suggested
Genetic and biochemical studies have established that palmitoylation of proteins on the cytoplasmic face of cell membranes is catalyzed by a family of integral membrane proteins with a conserved Asp-His-His-Cys (DHHC) motif embedded in a cysteine-rich domain
DHHC Proteins Oligomerize in Mammalian Cells—To determine whether DHHC proteins exist as dimers or higher-order oligomers in living cells, Bioluminescence resonance energy transfer (BRET) was used to detect homomeric interactions of DHHC palmitoyltransferases in living cells
Summary
Oligomerization of DHHC palmitoyltransferases has been suggested. Results: Self-association of DHHC2 and DHHC3 was detected in intact cells and in vitro. Genetic and biochemical studies have established that palmitoylation of proteins on the cytoplasmic face of cell membranes is catalyzed by a family of integral membrane proteins with a conserved Asp-His-His-Cys (DHHC) motif embedded in a cysteine-rich domain (reviewed in Ref. 1–3). These socalled DHHC proteins are present in all eukaryotes as multienyzme families that range in size from five members in Schizosaccharomyces pombe to 24 in mouse and Arabidopsis [4]. Our results suggest that DHHC proteins form oligomers in cell membranes and may exist in a monomer-dimer equilibrium in which the active enzyme favors the monomeric state
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