The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8

Abstract

Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3͞Ynl326c is required for efficient localization of Vac8 to vacuoles in vivo, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuoleassociated Vac8 appears to be palmitoylated in a pfa3 mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate͞palmitate (as in Vac8) but also one that is targeted by a myristate͞basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole.Yck3 ͉ SH4 domain ͉ acylation ͉ membrane targeting P rotein and lipid trafficking along the endomembrane system occurs by vesicular transport (1). Of all proteins implicated in vesicle fusion and fission, only a subset is permanently associated with membranes via transmembrane segments, whereas most are recruited to the membrane from the cytoplasm. The latter proteins depend on membrane receptors, lipids, or lipid anchors for binding to their appropriate target membrane (2). This poses the question of how the recruitment of these proteins is coordinated with their function.Palmitoylation has been discussed as a special lipid modification. It may direct proteins to specific membrane domains (3-5), and it is the only common lipid modification that is reversible, permitting cycling of a protein between membranes and cytosol. The identification and characterization of the underlying acylation͞deacylation machinery is therefore critical for understanding the function of palmitoylation. Recently, biochemical and genetic analyses have identified several proteins that are required for palmitoylation, including those of the so-called DHHC-CRD family of polytopic membrane proteins (6). Yeast contains seven homologs (Erf2, Swf1, Yol003c, Akr1, Akr2, Ydr459c, and Ynl326c͞Pfa3), which seem to be distributed throughout the endomembrane system as suggested by the GFP database and recent studies. At the ER, Swf1 is required for palmitoylation of Tlg1 (7), and Erf2 promotes Ras2 palmitoylation (8, 9). The Golgi-localized Akr1 is responsible for palmitoylation of the casein kinase I (CKI) isoform Yck2 (10). Similarly, several of the 20 or so mammalian DHHC proteins localize to distinct organelles, with critical roles for the palmitoylation of PSD-95, Ras, and SNAP-25 (11-13). This wide distribution of proteins involved in palmitoylation suggests that palmitoylati...

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

“…Whereas full-length myr-Vac8 was palmitoylated almost exclusively by Pfa3, the myr-Vac8 SH4 domain fused to GFP was palmitoylated by each of the five DHHC proteins tested. This is consistent with in vivo studies demonstrating that Vac8[SH4]-GFP is palmitoylated to similar levels in WT and pfa3⌬ cells (37). Additionally, WT Vac8-GFP localized exclusively to the limiting membrane of the vacuole, but Vac8[SH4]-GFP was found on both the plasma membrane and internal membranes (37).…”

“…Thus, there may be some DHHC redundancy in regard to Meh1 palmitoylation. This would explain results observed by Hou et al (37) showing that membrane association of Meh1 was unchanged in akr1⌬ cells, whereas mutation of cysteines 7 and 8 produced a soluble protein (43). Neither myr-Ygl108 nor myr-Meh1 was a good substrate for Pfa3, suggesting that Pfa3 does not universally recognize SH4 domain proteins as substrates.…”

“…Evidence suggests that Pfa3 is responsible for the majority of Vac8 palmitoylation (26,37). However, deletion of PFA3 does not eliminate palmitoylation of overexpressed Vac8 (26).…”

A Novel Subtype of AP-1-binding Motif within the Palmitoylated trans-Golgi Network/Endosomal Accessory Protein Gadkin/γ-BAR

“…Detection of Protein Palmitoylation Using the Biotin-switch Method-Protein palmitoylation was analyzed according to a previous study (20) using the biotin-switch method. FLAGGadkin-(1-140) was immunoprecipitated from transfected COS7 cells (see above) using anti-FLAG antibodies.…”

“…2D). Second, palmitoylation of Gadkin-(1-140) was analyzed using the biotin-switch method (20) in which free cysteines are first protected by N-ethylmaleimide, then palmitate is dissociated from palmitoylated cysteine residues by treatment with hydroxylamine (NH 2 OH) liberating selectively palmitoylated cysteines that are then cross-linked to biotin and thus can be decorated with streptavidin-HRP. In agreement with the data above, immunoprecipitated FLAG-Gadkin-(1-140) was detected with streptavidin-HRP following hydroxylamine cleavage but not after treatment of samples with Tris (Fig.…”