THE CYSTM DOMAIN IS BOUND TO MEMBRANES BY PALMITOYLATION

GONZALO BIGLIANI; MARIA LUZ GIOLITO; JAVIER VALDEZ TAUBAS

ON lINE

Congreso; LVII SAIB XVI SAMIGE; 2021

SAIB-SAMIGE

CYSTM proteins are a superfamily of proteins which were identified using bioinformatics approaches and found to be widelydistributed among Eukaryotes. These proteins are in general small, ranging from 60 to 120 amino acids. CYSTM proteins arecharacterized by the presence of a conserved motif at the C-terminal region, which is rich in cysteine residues and that hasbeen annotated as a transmembrane domain (TMD). Orthologues of the CYSTM proteins in different organisms are involvedin resistance to pathogens and they might be involved in resistance to different kinds of stress. However, no thoroughexperimental 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 aspalmitoylation, is a post-translational modification that consists of the addition of long-chain lipids on cysteine residues. Thismodification is mediated by a family of transmembrane enzymes called Palmitoyltransferases (PATs) and it plays multipleroles in the regulation of many biological processes. Here we characterized the CYSTM proteins from Saccharomycescerevisiae. 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 sevenyeast PATs showed that the half-live of these proteins is dependent on the Palmitoyltransferase Akr1 which is known to modifyperipheral membrane proteins. Degradation of Ybr016w is mediated by the Ubiquitin ligase Rsp5 but it does not take place inthe vacuole as is usual for transmembrane proteins that localized at the plasma membrane. ABE combined with PEGylationexperiments showed that at list four of the five cysteine residues of the CYSTM domain in Ybr016w are S-acylated. Pointmutation 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. Palmitoylationmostly occurs at the cytoplasm and only residues at the cytoplasmic border of a TMD can be modified. Finally, treatment ofcell extracts containing Ybr016w with hydroxylamine, which cleaves thioester bound palmitates, results in the partial partitionof this protein to the soluble fraction. Our data suggest that these proteins are not transmembrane proteins as previouslysuggested, but they are bound to the membrane via palmitates and that the CYSTM module is in fact a palmitoylated domain.