Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters

Bizzozero, Oscar A.; Bixler, Heather A.; Pastuszyn, Andrzej

doi:10.1016/s0167-4838(00)00291-0

Cited by 51 publications

(56 citation statements)

References 47 publications

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“…Because in vitro palmitoylation required equimolar amounts of Ykt6 to modify Vac8, we suggested that acylation might occur by a nonenzymatic transfer mechanism (3,19). Such a mechanism has been suggested before and would explain previous autoacylation data (31). Initial data on the biochemical properties of DHHC-CRD protein pointed to the key function of the DHHC motif in palmitoylation but also showed similar acyl transfer properties as observed for Ykt6 (8, 10).…”

Section: Discussionsupporting

confidence: 60%

“…Initial data on the biochemical properties of DHHC-CRD protein pointed to the key function of the DHHC motif in palmitoylation but also showed similar acyl transfer properties as observed for Ykt6 (8, 10). However, more recent studies on DHHC-dependent palmitoylation have demonstrated enzymatic activity with turnover numbers ranging between three per min for Ras (13) and two per h for Vac8 (31). This rate is still very poor for Vac8, suggesting that additional factors like Ykt6 and Sec18 increase efficiency of this reaction in vitro.…”

Section: Discussionmentioning

confidence: 99%

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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: 60%

Section: Discussionmentioning

confidence: 99%

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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“…Multiple sequence alignment of Bet3 sequences showed adjacent to the palmitoylated cysteine an arginine, which is completely conserved from yeast to man (8,9). Basic amino acids in the vicinity of palmitoylated cysteines often affect the acylation reaction, probably by decreasing the pK a of the cysteine's -sulfhydryl group (19,20). Replacing arginine-67 by glutamic acid almost completely blocked palmitoylation of Bet3 (Fig.…”

Section: Resultsmentioning

confidence: 90%

“…Acyl-chain transfer (either enzyme-mediated or chemical) occurs through nucleophilic attack of a cysteine in the substrate protein on the carbonyl of the COS bond in the Pal-CoA molecule (20). To act as good nucleophile the cysteine should be present in its deprotonated form.…”

Section: Resultsmentioning

confidence: 99%

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Unique self-palmitoylation activity of the transport protein particle component Bet3: A mechanism required for protein stability

Kümmel

Heinemann

Veit³

2006

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

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Bet3 is a component of the transport protein particle complex involved in vesicular trafficking to and through the Golgi complex. X-ray structural analysis of human and mouse Bet3 revealed a hydrophobic tunnel inside the protein, which is occupied by a fatty acid linked to cysteine-68. We show here that Bet3 has strong self-palmitoylating activity. Incubation of purified Bet3 with [ 3 H]palmitoyl-CoA (Pal-CoA) leads to a rapid and stoichiometric attachment of fatty acids to cysteine-68. Bet3 has an intrinsic affinity for Pal-CoA, and the palmitoylation reaction occurs at physiological pH values and Pal-CoA concentrations. Moreover, Bet3 is also efficiently palmitoylated at cysteine-68 inside vertebrate cells. Palmitoylation can occur late after Bet3 synthesis, but once the fatty acids are bound they are not removed, not even by disassembly of the Golgi complex. Narrowing the hydrophobic tunnel by exchange of alanine-82 with bulkier amino acids inhibits palmitoylation, both in vitro and inside cells, indicating that the fatty acid must insert into the tunnel for stable attachment. Finally, we show that palmitoylation of Bet3 plays a structural role. CD spectroscopy reveals that chemically deacylated Bet3 has a reduced melting temperature. As a consequence of its structural defect nonacylated Bet3 does not bind to TPC6, a further subunit of the transport protein particle complex, and is degraded inside cells.Golgi ͉ protein structure

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Proteomic analysis of protein S‐nitrosylation

Torta¹,

Usuelli

Malgaroli

et al. 2008

Proteomics

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Nitric oxide (NO) produces covalent PTMs of specific cysteine residues, a process known as S-nitrosylation. This route is dynamically regulated and is one of the major NO signalling pathways known to have strong and dynamic interactions with redox signalling. In agreement with this scenario, binding of NO to key cysteine groups can be linked to a broad range of physiological and pathological cellular events, such as smooth muscle relaxation, neurotransmission and neurodegeneration. The characterization of S-nitrosylated residues and the functional relevance of this protein modification are both essential information needed to understand the action of NO in living organisms. In this review, we focus on recent advances in this field and on state-of-the-art proteomic approaches which are aimed at characterizing the S-nitrosylome in different biological backgrounds.

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Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters

Cited by 51 publications

References 47 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

Unique self-palmitoylation activity of the transport protein particle component Bet3: A mechanism required for protein stability

Proteomic analysis of protein S‐nitrosylation

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