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REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM

脯氨酸代谢的调节和划分

基本信息

批准号：
6180105
负责人：
MARJORIE C BRANDRISS
金额：
$ 30.37万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-06-01 至 2002-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6180105
关键词：
Saccharomyces cerevisiae aminoacid metabolism biological signal transduction conformation intermolecular interaction mass spectrometry molecular cloning nitrogen fixation phosphorylation proline protein structure function transcription factor ubiquitin western blottings yeast two hybrid system

项目摘要

DESCRIPTION:The long term objective of this project is to understand molecular mechanisms that regulate gene expression in nitrogen assimilation, including the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. In the wild, proline is the predominant nitrogen source for this organism. Its utilization requires the exhaustion of preferred, but less abundant, nitrogen sources and induction of the specific genes that encode the enzymes of this pathway. Induction is mediated by the Put3 protein, a transcriptional regulator that sits poised at the promoters of its target genes, remaining inactive unless proline is present. The focus of this study is to determine how Put3p is converted from an "off" to an "on" state and how it senses that proline is present and preferred nitrogen sources are absent from the growth medium. Two general models will be tested: a protein-protein or intermolecular interaction model and a conformational change or intramolecular interaction model. Put3p is modified by phosphorylation and ubiquitination; the role of each in the activity of the protein will be tested. Wild-type, constitutive and non-inducible put3 mutants will be examined for differences in modification. Pure Put3p will be analyzed by mass spectrometry to identify sites of modification. Limited proteolysis will be employed to determine if Put3p undergoes conformational changes in the presence of proline. The effect of proline on activation of Put3p is an in vitro transcription system will be tested. Put3p-interacting proteins will be sought using two-hybrid selections employing fragments of Put3p as the bait, and by the isolation of suppressors of Put3p constitutive mutants. Intramolecular interactions will be examined by a directed two-hybrid approach using fragments of Put3p as both bait and prey. The roles of the five characterized nitrogen repression regulators in the proline utilization system will be studied. This system provides a useful model to understand how changes in the cellular environment are signaled to the elements of a metabolism in humans. The work in Saccharomyces cerevisiae on proline metabolism contributed significantly to the recent isolation of human genes encoding homologous enzymes. Deficiencies in these enzymes cause hyperprolinemias; studies on the human genes will lead to a greater understanding of the molecular basis of these diseases.

描述：这个项目的长期目标是理解