The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase.
Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.
Roth
Amy F
AF
Department of Surgery, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Feng
Ying
Y
Chen
Linyi
L
Davis
Nicholas G
NG
D000217:Acyltransferases; D000818:Animals; D017089:Ankyrin Repeat; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D019308:Palmitic Acid / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D010171:Palmitoyl Coenzyme A; D019308:Palmitic Acid; D014166:Transferases; D000217:Acyltransferases; C107846:AKR1 protein, S cerevisiae; D003545:Cysteine