The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

Smotrys, Jessica E.; Schoenfish, Marissa J.; Stutz, Monica A.; Linder, Maurine E.

doi:10.1083/jcb.200507048

Cited by 73 publications

(96 citation statements)

References 40 publications

(66 reference statements)

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“…Also, deletion of ERF2 still permits partial Ras2 palmitoylation and localization (33), and our data suggest that some Vac8 is palmitoylated in pfa3⌬ cells. In agreement with this postulate, Linder and colleagues (28) detected residual palmitoylation of Vac8 by metabolic labeling with [ 3 H]palmitate in pfa3⌬ cells. The localization of Vac8 to pfa3⌬ vacuoles is in striking contrast to the poor binding of a Vac8 mutant that lacks its N-terminal cysteines and thus cannot be palmitoylated (K.S., L.E.P.D., H.H., T.J.L., and C.U., unpublished work).…”

Section: Discussionsupporting

confidence: 55%

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

Hou

Subramanian

LaGrassa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionsupporting

confidence: 55%

“…Pfa3 is required for efficient Vac8 localization to vacuoles, consistent with its ability to promotes palmitoylation in vitro upon reconstitution (28). Loss of Pfa3 leads to partial mislocalization of Vac8 to the cytosol and a reduction in vacuole fusion.…”

Section: Discussionmentioning

confidence: 83%

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

Hou

Subramanian

LaGrassa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…For the quantitation presented in Fig. 2C, soluene extraction of the protein of interest was performed as described previously (26,29). For the quantitation presented in Figs.…”

Section: Methodsmentioning

confidence: 99%

“…The N-terminal cysteines, and presumably the palmitoylation that occurs there, are required for the function of Vac8 in vacuolar fusion and vacuolar inheritance (18,24,25). We recently demonstrated both genetically and biochemically that the yeast DHHC protein Pfa3 is a Vac8 PAT (26).…”

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confidence: 99%

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Nadolski¹,

Linder

2009

Journal of Biological Chemistry

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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 ...

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“…In contrast to other acyl transferases that are soluble, the DHHCs are multi-membrane-spanning domain-containing proteins with the catalytic motif facing the cytosol (Mitchell et al, 2006). Site-directed mutagenesis established a direct involvement of the DHHC motif in palmitate transfer, since substitution of the cysteine residue was enough to abolish both autopalmitoylation of the PAT and palmitoylation of substrates (Roth et al, 2002;Smotrys et al, 2005). It was recently clearly demonstrated that the auto-palmitoylated PAT serves as a covalent intermediate between the donor palmitoyl-CoA and the acceptor in a two-step process (Jennings and Linder, 2012).…”

Section: Enzymes Implicated In Protein Palmitoylationmentioning

confidence: 99%

Emerging roles for protein S-palmitoylation in Toxoplasma biology

Frénal

Kemp

Soldati‐Favre

2014

International Journal for Parasitology

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a b s t r a c tPost-translational modifications are refined, rapidly responsive and powerful ways to modulate protein function. Among post-translational modifications, acylation is now emerging as a widespread modification exploited by eukaryotes, bacteria and viruses to control biological processes. Protein palmitoylation involves the attachment of palmitic acid, also known as hexadecanoic acid, to cysteine residues of integral and peripheral membrane proteins and increases their affinity for membranes. Importantly, similar to phosphorylation, palmitoylation is reversible and is becoming recognised as instrumental for the regulation of protein function by modulating protein interactions, stability, folding, trafficking and signalling. Palmitoylation appears to play a central role in the biology of the Apicomplexa, regulating critical processes such as host cell invasion which is vital for parasite survival and dissemination. The recent identification of over 400 palmitoylated proteins in Plasmodium falciparum erythrocytic stages illustrates the broad spread and impact of this modification on parasite biology. The main enzymes responsible for protein palmitoylation are multi-membrane protein S-acyl transferases harbouring a catalytic Asp-HisHis-Cys (DHHC) motif. A global functional analysis of the repertoire of protein S-acyl transferases in Toxoplasma gondii and Plasmodium berghei has recently been performed. The essential nature of some of these enzymes illustrates the key roles played by this post-translational modification in the corresponding substrates implicated in fundamental processes such as parasite motility and organelle biogenesis. Toward a better understanding of the depalmitoylation event, a protein with palmitoyl protein thioesterase activity has been identified in T. gondii. TgPPT1/TgASH1 is the main target of specific acyl protein thioesterase inhibitors but is dispensable for parasite survival, suggesting the implication of other genes in depalmitoylation. Palmitoylation/depalmitoylation cycles are now emerging as potential novel regulatory networks and T. gondii represents a superb model organism in which to explore their significance.

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The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

Cited by 73 publications

References 40 publications

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

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

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Emerging roles for protein S-palmitoylation in Toxoplasma biology

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