PROTEOMIC ANALYSIS OF PROTEIN PALMITOYLATION IN YEAST

酵母蛋白质棕榈酰化的蛋白质组学分析

• 批准号: 7602208
• 负责人: NICHOLAS G DAVIS
• 金额: $ 0.62万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602208
• 关键词: Biotin; Biotinylation; Chemicals; Complex; Computer Retrieval of Information on Scientific Projects Database; Cysteine-Rich Domain; Drops; Enzymes; Ethylmaleimide; Funding; Fungal Genome; Genes; Grant; Hydroxylamine; Institution; Link; Mediating; Membrane Proteins; Methodology; Methods; Modification; Personal Satisfaction; Protein Analysis; Protein Family; Proteins; Proteome; Proteomics; Protocols documentation; Reagent; Research; Research Personnel; Resources; Source; Specificity; Sulfhydryl Compounds; Transferase; United States National Institutes of Health; Work; Yeasts; Zinc Fingers; palmitoylation

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.

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