Analysis of DHHC acyltransferases implies overlapping substrate specificity and a two-step reaction mechanism.
Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD) acyltransferases are polytopic transmembrane proteins that are found along the endomembrane system of eukaryotic cells and mediate palmitoylation of peripheral and integral membrane proteins. Here, we address the in vivo substrate specificity of five of the seven DHHC acyltransferases for peripheral membrane proteins by an overexpression approach. For all analysed DHHC proteins we detect strongly overlapping substrate specificity. In addition, we now show acyltransferase activity for Pfa5. More importantly, the DHHC protein Pfa3 is able to trap several substrates at the vacuole. For Pfa3 and its substrate Vac8, we can distinguish two consecutive steps in the acylation reaction: an initial binding that occurs independently of its central cysteine in the DHHC box, but requires myristoylation of its substrate Vac8, and a DHHC-motif dependent acylation. Our data also suggest that proteins can be palmitoylated on several organelles. Thus, the intracellular distribution of DHHC proteins provides an acyltransferase network, which may promote dynamic membrane association of substrate proteins.
Hou
Haitong
H
University of Osnabrück, Department of Biology, Biochemistry section, Osnabrück, Germany.
John Peter
Arun T
AT
Meiringer
Christoph
C
Subramanian
Kanagaraj
K
Ungermann
Christian
C
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D054878:Lipoylation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C111373:VAC8 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000217:Acyltransferases; C505807:Pfa3 protein, S cerevisiae; D047389:Casein Kinase I; C479666:YCK2 protein, S cerevisiae; C479667:YCK3 protein, S cerevisiae