PROTEOMIC ANALYSIS OF PROTEIN PALMITOYLATION IN YEAST

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们已经开发了一种蛋白质组学方案，允许从复杂的蛋白质提取物中纯化棕榈酰化的蛋白质子集。新方法的关键是生物素的化学交换，以获得附着的棕榈酰修饰。首先用NEM彻底处理萃取蛋白以阻断游离硫醇。接下来，附着的棕榈酰基与羟胺一起释放，暴露出新的硫醇。然后，用硫醇特异性生物素化试剂对新暴露的硫醇进行生物素化。这种方法效果很好，对棕榈酰化有很强的特异性。使用这种方法，第一个项目将是酵母棕榈酰蛋白质组的全面表征。第二个项目将把已鉴定的棕榈酰蛋白与介导其棕榈酰化的酶联系起来，即与它们的同源蛋白酰基转移酶（PAT）联系起来。我们之前的结果已经指向DHHC蛋白家族，一组具有锌指状DHHC半胱氨酸丰富结构域的多聚膜蛋白，可能是PAT特异性的一个家族。酵母基因组编码7种DHHC蛋白。单个或组合删除不同DHHC基因的菌株将按照上述方法对其棕榈酰蛋白质组进行分析。从特定DHHC缺失菌株谱中脱落的棕榈酰蛋白将进一步被表征为缺失的PAT酶的潜在底物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a proteomic protocol that allows the purification of the palmitoylated subset of proteins from complex protein extracts. The crux of the new methodology is a chemical exchange of biotin for the attached palmitoyl modifications. Extract proteins are first treated exhaustively with NEM to block free thiols. Next, the attached palmitoyl groups are released with hydroxylamine, exposing new thiols. Then, the newly-exposed thiols are biotinylated with a thiol-specific biotinylation reagent. The method works well, being quite specific for palmitoylation. Using this methodology, a first project will be a comprehensive characterization of the yeast palmitoyl-proteome. A second project will link identified palmitoyl-proteins to the enzymes that mediate their palmitoylation, that is to their cognate protein acyl transferase (PAT). Our prior results have pointed towards the DHHC protein family, a set of polytopic membrane proteins with zinc finger-like DHHC cysteine-rich domains, as likely being a family of PAT specificities. The yeast genome encodes seven DHHC proteins. Strains deleted for different DHHC genes either singly or in combination will have their palmitoyl-proteomes profiled as described above. Palmitoyl-proteins dropping out of particular DHHC deletion strains profiles will be further characterized as potential substrates for the deleted PAT enzyme.