PROTEOMIC ANALYSIS OF PROTEIN PALMITOYLATION IN YEAST

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经开发了一种蛋白质组学协议，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与附着的棕榈酰修饰的化学交换。 提取物蛋白质首先用NEM彻底处理以封闭游离巯基。 接下来，连接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将新暴露的硫醇生物素化。 该方法效果良好，对棕榈酰化相当特异。 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。 第二个项目将确定棕榈酰蛋白质与介导其棕榈酰化的酶，即与其同源蛋白质酰基转移酶（PAT）联系起来。 我们先前的结果已经指向DHHC蛋白家族，一组具有锌指样DHHC富含半胱氨酸结构域的多位膜蛋白，可能是PAT特异性的家族。 酵母基因组编码七种DHHC蛋白。 单独或组合缺失不同DHHC基因的菌株将具有如上所述的棕榈酰-蛋白质组谱。 从特定DHHC缺失菌株谱中脱落的棕榈酰蛋白将进一步表征为缺失的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.