PALMITOYLATION, STABILITY AND FUNCTION OF THE CYSTM FAMILY OF PROTEINS IN YEAST.

MARIA LUZ GIOLITO; GONZALO BIGLIANI; JAVIER VALDEZ TAUBAS

Mendoza

Congreso; 58 SAIB; 2022

SAIB-SAMIGE

CYSTM proteins are a superfamily of proteins widely distributed among Eukaryotes. These proteins are small, ranging from 60 to120 amino acids and characterized by the presence of a conserved motif at the C-terminal region, which is rich in cysteine residuesand has been annotated as a transmembrane domain (TMD). In the yeast Saccharomyces cerevisiae, the family has no knownfunctions and comprises the genes YBR016W, YDL012C, YDR034W-B, YDR210W and possibly MNC1 (YBR056W-A). Someplant homologues of these proteins have been linked to resistance to pathogens and also to abiotic stresses, but no mechanisticexplanations for these phenotypes have been proposed.Protein S-acylation, commonly known as palmitoylation, is a highly prevalent, post-translational modification (PTM) that consistsof the addition of long-chain lipids onto cysteine residues. This PTM is mediated by a family of Palmitoyltransferases (PATs) andplays multiple roles in important biological processes. We have suggested that CYSTM proteins do not possess a TMD aspostulated, but are bound to membranes by palmitoylation of the cysteines in the CYSTM domain. Here, present additionalevidence for this claim, which includes extensive mutagenesis of the CYSTM domain, followed by fluorescence microscopy,subcellular fractionation and biochemical determination of their palmitoylation status. These experiments allow us to postulate amodel for how CYSTM domains interact with membranes. Expression of Ybr016w in strains lacking each of the seven PATsindicated that this protein is completely degraded, in a proteasome-dependent manner, in the absence of the PAT Akr1, and to abouthalf of its steady-state levels, in the absence of Erf2. We generated a series of constructs in which the CYSTM domain of YBR016wwas either deleted, replaced by the transmembrane domain of Sso1 or mutated so that all cysteines were replaced by Alanines.Analyses of these constructs in wt, akr1Δ and erf2Δ strains indicated that the stability of Ybr016w is affected by the lack of Erf2-mediated palmitoylation. Interestingly, the complete degradation of YBR016W observed in akr1Δ strains is independent of theCYSTM domain. Finally, we began to explore the possible functions of this family of proteins. Growth tests in different mediashowed that the lack of these genes makes strains more resistant to the presence of 6 mM Zn2+ and to a lesser extent to the presenceof 8 mM Mn2+. Other bivalent cations such as Cd2+ or Cu2+ did not elicit growth differences with respect to the wt.Finally, we are carrying out proximity labelling experiments (Turbo-ID) to uncover interacting partners of the CYSTM family ofproteins by LC followed by mass spectrometry. We expect that these experiments will lead to a better understanding of this proteinfamily.