PALMITOYLATION OF THE CYSTM FAMILY OF PROTEINS IN YEAST

Salta

Congreso; LV Annual Meeting of the Argentine Society of Research in Biochemistry and Molecular Biology. (SAIB) and XIV Pan American Societies of Biochemistry and Molecular Biology Congress.; 2019

A superfamily of proteins called CYSTM proteins was identified using a bioinformatics approach and found to be widely distributed amongeukaryotes. These proteins are in general small, ranging from 60 to 120 aminoacids. The family is characterized by the presence of a conservedmotif at the C-terminal region, which is rich in cysteine and that has been annotated as a transmembrane helix. High-throughput studies suggestthat members of this family are localized to the plasma membrane. Orthologues of these proteins are involved in resistance to pathogens and theymight be involved in resistance to different kinds of stress, including that caused by heavy metals. However, no thorough experimental analysis ofthis family of proteins has been carried out. In yeast, the family comprises the genes YDL012C, YBR016W, YDR034W-B, YDR210W and therecently characterized Mnc1 (YBR056W-A) Manganese-chelating protein 1. We became interested in these proteins because the CYSTM modulecould be the substrate of palmitoylation and if so, might not be able to form a TMD as predicted. Moreover, YBR016W was suggested to bepalmitoylated in a high-throughput study. Protein S-acylation, commonly known as palmitoylation, is a post-translational modification (PTM) thatconsists of the addition of long-chain lipids on cysteine residues. This modification plays some critical roles in the regulation of many cellularprocesses. Palmitoylation is mediated by a family of transmembrane palmitoyltransferases (PATs), which are defined by the presence of aconserved Asp-His-His-Cys (DHHC) catalytic domain. Saccharomyces cerevisiae has seven members of this family in its genome. YBR016W,YDR034W-B, YDR210W were fused to GFP and we confirmed that they are indeed localized to the plasma membrane, although in polarizedfashion. This polarity is achieved by endocytic cycling since it is lost in the endocytosis mutant sla1Δ and the proteins are retained in innerstructures in a recycling mutant ric1Δ. Acyl-biotin exchange (ABE) experiments indicate that these proteins are indeed palmitoylated. Expressionof YBR016W in strains lacking each of the yeast PATs showed that the plasma membrane localization, and most of the fluorescence, is lost in thestrain that lacks the PAT Akr1. This degradation was confirmed by Western blot. ABE-PEG indicates that although the protein is degraded,palmitoylation is not completely lost suggesting that other PAT must be modifying it. Finally, treatment with the palmitoylation inhibitor 2-bromopalmitate results in loss of fluorescence from the plasma membrane, suggesting that these proteins are indeed bound to the membrane viapalmitates and that the CYSTM module is, in fact, a palmitoylation motif.