A Plant Protein Kinase and A Phosphatidylinositol 3-Kinase Involved in Vacuolar Protein Transport and Development

96-30108 DeWald植物细胞液泡的功能类似于真菌的液泡和哺乳动物细胞的溶酶体。此外，植物细胞液泡还有其他功能：它们调节细胞液的pH值、渗透压休克和蛋白质的储存。植物液泡可以占据细胞总体积的90%。液泡的维持需要蛋白质和脂类从它们的合成部位到液泡的特定分类。蛋白质的识别、分选和运输到液泡的过程是复杂的。已有30多个基因参与了空泡蛋白的分选。本研究的目的是确定磷脂酰肌醇3-激酶(PI-3-Kinase)和丝氨酸/苏氨酸蛋白激酶在植物细胞液泡蛋白质分选中的作用。本研究的具体目的是：1)VPS 34P PI 3-K活性在拟南芥植物液泡蛋白分选中的作用；2)VSP 15P蛋白在拟南芥植物液泡蛋白分选中的作用。VPS 15P的基因将被克隆和测序。该基因的克隆将通过利用A.thaliana cDNA文库转化Vps 15p酵母突变株的功能互补来完成，或者通过基于PCR的基因片段的扩增来完成，这些基因片段将被用来探测cDNA文库以鉴定基因。VPS 15P和VPS 34P的作用将通过检测野生型植物中的组织和时间基因表达模式，以及通过记录转基因植物中过表达该基因的影响来评估。预计这些数据将为植物中蛋白质运输是如何调控的提供新的见解。植物细胞液泡的功能类似于真菌的液泡和哺乳动物细胞的溶酶体。此外，植物细胞液泡还有其他功能：它们调节细胞液的pH值、渗透压休克和蛋白质的储存。植物液泡可占细胞总体积的90%。液泡的维持需要蛋白质和脂类从它们的合成部位到液泡的特定分类。蛋白质的识别、分选和运输到液泡的过程是复杂的。已有30多个基因参与了空泡蛋白的分选。本研究的目的是确定磷脂酰肌醇3-激酶(PI-3-Kinase)和丝氨酸/苏氨酸蛋白激酶在植物细胞液泡蛋白质分选中的作用。本研究的具体目的是：1)VPS 34P PI 3-K活性在拟南芥植物液泡蛋白分选中的作用；2)VSP 15P蛋白在拟南芥植物液泡蛋白分选中的作用。VPS 15P的基因将被克隆和测序。该基因的克隆将通过利用A.thaliana cDNA文库转化Vps 15p酵母突变株的功能互补来完成，或者通过基于PCR的基因片段的扩增来完成，这些基因片段将被用来探测cDNA文库以鉴定基因。VPS 15P和VPS 34P的作用将通过检测野生型植物中的组织和时间基因表达模式，以及通过记录转基因植物中过表达该基因的影响来评估。预计这些数据将为植物中蛋白质运输是如何调控的提供新的见解。***

96-30108 DeWald The plant cell vacuole is functionally similar to fungi vacuoles and to lysosome of mammalian cells. In addition, plant cell vacuoles have other functions; they function in regulating cytosolic pH, osmotic shock, and in the storage of proteins. Plant vacuole can occupy up to 90% of the total cellular volume. The maintenance of vacuoles requires the specific sorting of proteins and lipids from their sites of synthesis to the vacuoles. The process of recognition, sorting, and transport of proteins to the vacuole is complex. More than 30 genes have been implicated in vacuolar protein sorting. The goal of the proposed research is to determine the role of a phosphatidylinositol 3-kinase (PI 3- kinase) and that of a serine/threonine protein kinase in protein sorting to plant cell vacuole. The specific objectives are to study 1) the role of Vps 34p PI 3-kinase activity in protein sorting to plant vacuoles in Arabidopsis thaliana; 2) the role of Vsp 15p, a protein kinase, in protein sorting to plant vacuole in A. thaliana. The gene for Vps 15p will be cloned and sequence. Cloning of the gene will be done by functional complementation of a Vps 15p yeast mutant by transforming with an A. thaliana cDNA library, or by PCR-based amplification of gene fragments that will be used to probe the cDNA library to identify the gene. The role of Vps 15p and of Vps 34p will be assessed in A. thaliana by examining tissue and temporal gene expression patterns in wild type plants, and by documenting the effects of overexpressing the gene in transgenic plants. It is expected that this data will provide new insights into how protein transport is regulated in plants. %%% The plant cell vacuole is functionally similar to fungi vacuoles and to lysosome of mammalian cells. In addition, plant cell vacuoles have other functions; they function in regulating cytosolic pH, osmotic shock, and in the storage of proteins. Plant vacuole can occupy up to 90% of the total cellular vo lume. The maintenance of vacuoles requires the specific sorting of proteins and lipids from their sites of synthesis to the vacuoles. The process of recognition, sorting, and transport of proteins to the vacuole is complex. More than 30 genes have been implicated in vacuolar protein sorting. The goal of the proposed research is to determine the role of a phosphatidylinositol 3-kinase (PI 3- kinase) and that of a serine/threonine protein kinase in protein sorting to plant cell vacuole. The specific objectives are to study 1) the role of Vps 34p PI 3-kinase activity in protein sorting to plant vacuoles in Arabidopsis thaliana; 2) the role of Vsp 15p, a protein kinase, in protein sorting to plant vacuole in A. thaliana. The gene for Vps 15p will be cloned and sequence. Cloning of the gene will be done by functional complementation of a Vps 15p yeast mutant by transforming with an A. thaliana cDNA library, or by PCR-based amplification of gene fragments that will be used to probe the cDNA library to identify the gene. The role of Vps 15p and of Vps 34p will be assessed in A. thaliana by examining tissue and temporal gene expression patterns in wild type plants, and by documenting the effects of overexpressing the gene in transgenic plants. It is expected that this data will provide new insights into how protein transport is regulated in plants. ***