PROTEOMIC ANALYSIS OF PROTEIN PALMITOYLATION IN YEAST

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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与棕榈酰化修饰的化学交换。提取的蛋白质首先用NEM彻底处理，以阻止游离硫醇。接下来，结合的棕榈酰基与羟胺一起释放，暴露出新的硫醇。然后，用硫醇特异的生物素化试剂将新暴露的硫醇生物素化。该方法效果良好，对棕榈酰化反应非常特异。使用这种方法，第一个项目将是酵母棕榈酰基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质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。我们先前的结果指向DHHC蛋白家族，这是一组具有锌指状DHHC富含半胱氨酸结构域的多表位膜蛋白，很可能是PAT特异性家族。酵母基因组编码七种DHHC蛋白。不同DHHC基因缺失的菌株，无论是单独删除还是组合删除，其棕榈酰基蛋白质组都将如上所述。从特定的DHHC缺失菌株中掉出的棕榈酰基蛋白质图谱将进一步被表征为缺失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.