Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Abstract

Palmitoylation of the yeast vacuolar protein Vac8 is important for its role in membrane-mediated events such as vacuole fusion. It has been established both in vivo and in vitro that Vac8 is palmitoylated by the Asp-His-His-Cys (DHHC) protein Pfa3. However, the determinants of Vac8 critical for recognition by Pfa3 have yet to be elucidated. This is of particular importance because of the lack of a consensus sequence for palmitoylation. Here we show that Pfa3 was capable of palmitoylating each of the three N-terminal cysteines of Vac8 and that this reaction was most efficient when Vac8 is N-myristoylated. Additionally, when we analyzed the Src homology 4 (SH4) domain of Vac8 independent of the rest of the protein, palmitoylation by Pfa3 still occurred. However, the specificity of palmitoylation seen for the full-length protein was lost, and the SH4 domain was palmitoylated by all five of the yeast DHHC proteins tested. These data suggested that a region of the protein C-terminal to the SH4 domain was important for conferring specificity of palmitoylation. This was confirmed by use of a chimeric protein in which the SH4 domain of Vac8 was swapped for that of Meh1, another palmitoylated and N-myristoylated protein in yeast. In this case we saw specificity mimic that of wild type Vac8. Competition experiments revealed that the 11th armadillo repeat of Vac8 is an important element for recognition by Pfa3. This demonstrates that regions distant from the palmitoylated cysteines are important for recognition by DHHC proteins.S-Palmitoylation (hereafter referred to as palmitoylation) is a widespread post-translational modification in which the fatty acid palmitate (16:0) is attached to a cysteine residue via a thioester linkage. Palmitate can be released from a cysteine by hydrolysis of the thioester linkage; thus palmitoylation is a reversible modification. Numerous eukaryotic proteins are palmitoylated, and substrates include both peripheral and transmembrane domain proteins. The functional consequences of palmitoylation are diverse. Membrane association, protein stability, and protein trafficking are among the properties that can be modulated by the palmitoylation status of a protein (reviewed in 1, 2).The enzymes responsible for palmitoylation have only recently been identified. The first protein acyltransferases (PATs) 3 discovered were Erf2 and Akr1 in yeast, which palmitoylate Ras2 and Yck2, respectively (3, 4). Both Erf2 and Akr1 are transmembrane proteins with an Asp-His-(His/Tyr)-Cys (DHHC) motif embedded in a cysteine-rich domain (CRD). Homology with this domain has identified five additional DHHC proteins in Saccharomyces cerevisiae. These seven proteins account for the bulk of palmitoylating activity in the cell (5).To date 23 DHHC proteins have been identified in mammals (6). As the field has progressed DHHC proteins have been linked to the regulation of neurotransmission (7-9), nitric oxide release (10), and cell-cell contacts (11). Palmitoylation of Huntingtin by HIP14 (DHHC17) plays a protective role in ...

Substrate recognition by the cell surface palmitoyl transferase DHHC5

“…In some isoforms the region of C-tail disorder is relatively large (for example, DHHCs 1,5,8,and 14). Our experimental data together with these disorder predictions are consistent with previous reports that regions outside the cysteinerich domain are responsible for substrate recruitment to DHHCs (27)(28)(29). We suggest that disordered domain interactions underlie substrate recognition by (and hence substrate specificity of) some DHHCs.…”

Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole

“…Env7 contains a consensus sequence for S-palmitoylation in an N-terminal string of three cysteine residues, Cys13 to -15. Palmitoylation is a common lipidation that increases the affinity of a soluble protein for the cytoplasmic faces of membranes and affects protein localization and function (49). Since Env7 does not exhibit putative signal sequence or transmembrane domains when subjected to Kyte-Doolittle analysis (data not shown), palmitoylation could confer its vacuolar membrane association.…”

S-acylation-dependent association of the calcium sensor CBL2 with the vacuolar membrane is essential for proper abscisic acid responses

“…Vacuolar targeting of Vac8p from yeast is brought about by dual lipid modifications through myristoylation and S-acylation and myristoylation has been shown to be required for efficient S-acylation [40,42,45]. While S-acylation is required for proper Vac8p function in vacuole fusion it appears to be dispensable for vacuolar targeting.…”

“…Based on these findings and the cytoplasmic accumulation of CBL2 after application of 2-Br, we hypothesize that recruitment of CBL2 to the vacuolar membrane occurs by a direct cytoplasm to vacuolar membrane switch that may involve specific interaction with a PAT that resides in this membrane. While in mammalian cells none of the 23 different PATs appears to be localized to the lysosomal compartment [39], Pfa3p, one of the 7 PATs encoded in the yeast genome, has been identified as being localized in the vacuolar membrane [40], where this enzyme S-acylates the armadillo repeat protein Vac8p that is essential for vacuole fusion [40][41][42]. Similar to the situation in yeast at least one of the 23 PATs encoded in the genome of the plant Arabidopsis thaliana seems to be localized to the vacuolar membrane [43].…”