Transmembrane topology of the protein palmitoyl transferase Akr1.
The two recently identified protein acyl transferases (PATs), Akr1p and Erf2p/Erf4p, point toward the DHHC protein family as a likely PAT family. The DHHC protein family, defined by the novel, zinc finger-like DHHC cysteine-rich domain (DHHC-CRD), is a diverse collection of polytopic membrane proteins extending through all eukaryotes. To define the PAT domains that are oriented to the cytoplasm and are thus available to effect the cytoplasmically limited palmitoyl modification, we have determined the transmembrane topology of the yeast PAT Akr1p. Portions of the yeast protein invertase (Suc2p) were inserted in-frame at 10 different hydrophilic sites within the Akr1 polypeptide. Three of the Akr1-Suc2-Akr1 insertion proteins were found to be extensively glycosylated, indicating that the invertase segment inserted at these Akr1p sites is luminally oriented. The remaining seven insertion proteins were not glycosylated, consistent with a cytoplasmic orientation for these sites. The results support a model in which the Akr1 polypeptide crosses the bilayer six times with the bulk of its hydrophilic domains disposed toward the cytoplasm. Cytoplasmic domains include both the relatively large, ankyrin repeat-containing N-terminal domain and the DHHC-CRD, which maps to a cytosolic loop segment. Functionality of the different Akr1-Suc2-Akr1 proteins also was examined. Insertions at only 4 of the 10 sites were found to disrupt Akr1p function. Interestingly, these four sites all map cytoplasmically, suggesting key roles for these cytoplasmic domains in Akr1 PAT function. Finally, extrapolating from the Akr1p topology, topology models are proposed for other DHHC protein family members.
Politis
Eugenia G
EG
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Roth
Amy F
AF
Davis
Nicholas G
NG
D000217:Acyltransferases; D000483:Alleles; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005816:Genetic Complementation Test; D005838:Genotype; D006031:Glycosylation; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000737:chemistry
D008051:Lipid Bilayers; D029701:Saccharomyces cerevisiae Proteins; D000217:Acyltransferases; C107846:AKR1 protein, S cerevisiae; C468437:SUC2 protein, S cerevisiae; D043324:beta-Fructofuranosidase