?The CYSTM domain is bound to membranes by palmitoylation"

BIGLIANI, GONZALO; GIOLITO, MARIA LUZ; VALDEZ TAUBAS, JAVIER

Congreso; SAIB; 2021

CYSTM proteins are a superfamily of proteins which were identified using bioinformatics approaches and found to be widely distributed among Eukaryotes. These proteins are in general small, ranging from 60 to 120 amino acids. CYSTM proteins are characterized by the presence of a conserved motif at the C-terminal region, which is rich in cysteine residues and that has been annotated as a transmembrane domain (TMD). Orthologues of the CYSTM proteins in different organisms are involved in resistance to pathogens and they might be involved in resistance to different kinds of stress. However, no thorough experimental studies on this family of proteins have been carried out. In yeast, the family comprises the genes YBR016W, YDL012C, YDR034W-B, YDR210W and the recently characterized Manganese-chelating protein 1 (MNC1/YBR056W-A). Ybr016w was suggested to be palmitoylated in a high-throughput study. Protein S-acylation, commonly known as palmitoylation, is a post-translational modification that consists of the addition of long-chain lipids on cysteine residues. This modification is mediated by a family of transmembrane enzymes called Palmitoyltransferases (PATs) and it plays multiple roles in the regulation of many biological processes. Here we characterized the CYSTM proteins from Saccharomyces cerevisiae. Using confocal microscopy, we confirmed that members of these family localize to the plasma membrane. Particularly, Ybr016w displays a polarized distribution achieved by endocytic cycling. Acyl-biotin exchange (ABE) experiments indicate that CYSTM proteins are palmitoylated. Expression of Ybr016w in strains lacking each of the seven yeast PATs showed that the half-live of these proteins is dependent on the Palmitoyltransferase Akr1 which is known to modify peripheral membrane proteins. Degradation of Ybr016w is mediated by the Ubiquitin ligase Rsp5 but it does not take place in the vacuole as is usual for transmembrane proteins that localized at the plasma membrane. ABE combined with PEGylation experiments showed that at list four of the five cysteine residues of the CYSTM domain in Ybr016w are S-acylated. Point mutation of candidate cysteines indicate the cysteines located at the C-terminal region of CYSTM domain are palmitoylated, which, if the CYSTM domain was a TMD, would place them embedded in the exoplasmic leaflet of the bilayer. Palmitoylation mostly occurs at the cytoplasm and only residues at the cytoplasmic border of a TMD can be modified. Finally, treatment of cell extracts containing Ybr016w with hydroxylamine, which cleaves thioester bound palmitates, results in the partial partition of this protein to the soluble fraction. Our data suggest that these proteins are not transmembrane proteins as previously suggested, but they are bound to the membrane via palmitates and that the CYSTM module is in fact a palmitoylated domain.