Palmitoylation of the CYSTM family of proteins in yeast

MARIA LUZ GIOLITO; GONZALO BIGLIANI; JAVIER VALDEZ TAUBAS

Salta

Congreso; 55 SAIB; 2019

SAIB

A superfamily of proteins called CYSTM proteins was identified using a bioinformatics approach and found to be widely distributed across eukaryotes. These proteins are in general small, ranging from 60 to 120 aa. The family is characterised by the presence of a conserved motif at the C-terminal region, which is rich in cysteines and that has been annotated as a transmembrane helix. High-throughput studies suggest that members of this family are localised to the plasma membrane. Orthologues of these proteins are involved in resistance to pathogens and they might be involved in resistance to different kinds of stress, including that caused by heavy metals. However, no thorough experimental analyses of this family of proteins have been carried out. In yeast, the family comprises the genes YDL012C, YBR016W, YDR034W-B, YDR210W and the recently characterised Mnc1 (YBR056W-A) Manganese chelating protein 1.We became interested in these proteins because the CYSTM module could be the substrate of palmitoylation and if so, might not be able to form a TMD as predicted. Moreover, YBR016W was suggested to be palmitoylated in a high-throughput study. Protein S-acylation, commonly known as palmitoylation, is a post-translational modification (PTM) that consists in the addition of long-chain lipids on cysteine residues. This modification plays critical roles in the regulation of many cellular processes. Palmitoylation is mediated by a family of transmembrane palmitoyltransferases (PATs), which are defined by the presence of a conserved 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 localised to the plasma membrane, although in polarised fashion. This polarity is achieved by endocytic cycling since it is lost in the endocytosis mutant sla1Δ and the proteins are retained in inner structures in a recycling mutant ric1Δ. Acyl-biotin exchange (ABE) experiments indicate that these proteins are indeed palmitoylated. Expression of YBR016W in strains lacking each of the yeast PATs showed that the plasma membrane localization, and most of the fluorescence, is lost in the strain 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 via palmitates and that the CYSTM module is, in fact, a palmitoylation motif.