Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8

Abstract

Palmitoylation is a posttranslational modification that regulates protein trafficking and stability. In this study we investigated whether the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins syntaxin 7 and syntaxin 8 are modified with palmitate. Using metabolic labeling and sitedirected mutagenesis, we show that human syntaxins 7 and 8 are modified with palmitate through a thioester linkage. Palmitoylation is dependent upon cysteine 239 of human syntaxin 7 and cysteine 214 of syntaxin 8, residues that are located on the cytoplasmic face of the transmembrane domain (TMD). Palmitoylation of syntaxin 8 is minimally affected by the Golgi-disturbing agent brefeldin A (BFA), whereas BFA dramatically inhibits palmitoylation of syntaxin7. The differential effect of BFA suggests that palmitoylation of syntaxins 7 and 8 occurs in distinct subcellular compartments. Palmitoylation does not affect the rate of protein turnover of syntaxins 7 and 8 nor does it influence the steady-state localization of syntaxin 8 in late endosomes. Syntaxin 7 actively cycles between endosomes and the plasma membrane. Palmitoylation-defective syntaxin 7 is selectively retained on the plasma membrane, suggesting that palmitoylation is important for intercompartmental transport of syntaxin 7.-He, Y., and M. E. Linder. Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8. J. Lipid Res. 2009. 50: 398-404. Supplementary key words protein trafficking • fatty acylation • brefeldin A Palmitoylation refers to the posttranslational addition of palmitate to cysteine residues in proteins (1-3). Two features of palmitoylation distinguish it from other covalent lipid modifications that occur in the cytoplasm or on the cytoplasmic face of membranes. First, palmitoylation is reversible. Palmitate is added to proteins through a reversible thioester linkage, whereas myristate and prenyl groups are attached through stable amide and thioether linkages, respectively. Second, palmitoylation is a modification of both integral and peripheral membrane proteins, whereas Nmyristoylation and prenylation almost exclusively are modifications of proteins peripherally associated with membranes.A key function for lipid attachment to otherwise soluble proteins is to promote their interactions with membranes. Integral membrane proteins are permanently inserted into membranes by virtue of their transmembrane domains (TMDs). For many receptors and ion channels, palmitoylation of cytoplasmic tails creates additional sites of protein-membrane attachment (3). In other integral membrane proteins, however, the site of palmitoylation is often at one or more cysteine residues near the interface of the cytoplasm and the membrane. The functional consequences of palmitoylating integral membrane proteins are unclear; although evidence is emerging that palmitoylation may impact protein stability (4, 5).Several members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) fami...

Gi/o Signaling and the Palmitoyltransferase DHHC2 Regulate Palmitate Cycling and Shuttling of RGS7 Family-binding Protein

“…pTR-UF-tdRFP-R7BP was made by inserting the wild-type R7BP coding region into pTR-UF-tdRFP. Plasmids expressing GFP-N-Ras, GFP-Syntaxin 7, constitutively active G o ␣Q204L, pEGFP-R7BP, and p3xFLAG-R7BP clones have been described previously (23,31,32).…”

Cytosol dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly

“…Most SNAREs are transmembrane proteins but some of the SNAREs lack transmembrane domains and shuttle between cytosolic and membrane-bound forms. 22,23 Taken together, our data suggest that interphase cytosol contains fusogenic proteins that can be tightly associated with membranes but do not have transmembrane domains and can replace impaired or, in the case of liposomes, missing fusogenic proteins with intact copies of the same proteins.…”

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

“…Here, we show that in the absence of its TMD, Gos28 is still capable of facilitating membrane fusion, likely via its interaction with other membrane-bound SNARE proteins. Indeed, several SNARE proteins are attached to the membrane by post-translational modifications, such as prenylation and/or palmitoylation (13)(14)(15)(16)(17)(81)(82)(83). However, Gos28 has no consensus sequences for prenylation, palmitoylation, or any other lipid modifications such as myristoylation or glycosylphosphatidylinositol anchor attachment.…”

“…However, Gos28 has no consensus sequences for prenylation, palmitoylation, or any other lipid modifications such as myristoylation or glycosylphosphatidylinositol anchor attachment. Although the amino acid sequence requirements for palmitoylation are not well defined, it has been thoroughly established that the acylated cysteine residue is always located at the C terminus of the SNARE motif, either in place of a TMD, within a TMD, or immediately adjacent to a TMD (13)(14)(15)(81)(82)(83). The only cysteine residue that remains on the Gos28 ϪTMD protein is located at the N terminus of the SNARE motif (Fig.…”