Vacuolar Protein Sorting in Plant Cells

[9315940] Chrispeels, Marten J.该提案的目标是了解蛋白质如何被靶向并在植物细胞的液泡中积累。虽然植物液泡与哺乳动物细胞的酵母液泡和溶酶体有关，但它们含有更多种类的蛋白质，包括储存蛋白、凝集素、酶抑制剂、防御酶，以及所有液泡和溶酶体中常见的许多酸性水解酶。在过去的几年里，在理解空泡蛋白本身发生的分选信号方面取得了很大的进展，但目前还没有关于细胞机制的信息，使这些蛋白能够正确地靶向到它们的目的地。高尔基衍生囊泡的正确液泡靶向涉及囊泡外部的蛋白质，这些蛋白质以高度特异性的方式介导囊泡出芽、运输和囊泡与靶膜的融合。这些过程被认为需要用结合在膜上的蛋白质包衣和脱衣，这些蛋白质可以从细胞质溶胶中招募。鉴定这些蛋白质是本提案的主要目标。该项目将利用最近从拟南芥中分离到的编码磷脂酰肌醇3-激酶的基因，研究它是否在液泡蛋白分选中起作用。该蛋白的功能将在稳定转化和瞬态系统中或用反义寡脱氧核糖核苷酸处理的原生质体中进行检测。Pi将用植物磷脂酰肌醇3-激酶基因转化酵母vps34突变体，找到相互作用的蛋白，然后用植物cDNA文库拯救该菌株。双杂交方法也将用于鉴定该基因。Pi还将继续研究将蛋白质靶向到液泡膜或张力质体上。他将构建包含他有抗血清的报告蛋白和不同的膜跨越域的结构体。这项工作的意义在于更好地了解植物细胞中的液泡生物发生。这里提出的研究将有助于我们了解液泡蛋白到达目的地的机制。与分泌系统的其他方面一样，一些发现将适用于所有真核细胞，而另一些发现将适用于植物细胞。＊＊＊

9315940 Chrispeels, Marten J. The goal of the proposal is to understand how proteins are targeted to and accumulated in vacuoles of plant cells. Although plant vacuoles are related to yeast vacuoles and lysosomes of mammalian cells, they contain a much greater variety of proteins, including storage proteins, lectins, enzyme inhibitors, defense enzymes, as well as many of the acidic hydrolases commonly found in all vacuoles and lysosomes. Much progress has been made in the last years in understanding the sorting signals that occur on the vacuolar proteins themselves, but there is at present no information on the cellular machinery that allows these proteins to be correctly targeted to their destination. Correct vacuolar targeting of Golgi-derived vesicles involves proteins on the outside of the vesicles that mediate vesicle budding, transport and fusion of the vesicles with the target membrane in a highly specific manner. These processes are thought to require coating and uncoating with proteins that bind to the membranes and can be recruited from the cytosol. The identification of these proteins is the major goal of this proposal. The Pi will exploit the recent isolation of the gene that encodes a phosphatidyl-inositol 3-kinase from Arabidopsis and investigate whether it has a role in vacuolar protein sorting. The function of this protein will be examined in stably transformed and transient systems, or protoplasts treated with antisense oligodeoxyribonucleotides. The Pi will transform a yeast vps34 mutant with the plant phosphatidylinositol 3-kinase gene to find interacting proteins, and then rescue this strain with a plant cDNA library. The two-hybrid method is also going to be used to identify this gene. The Pi will also continue work on the targeting of proteins to the vacuolar membrane or tonoplast. He will make constructs that involve a reporter protein against which he has antiserum, and different membrane spanning domains. %%% The significance of this work lies in a better understanding of vacuole biogenesis in plant cells. The research proposed here will contribute to our understanding of the mechanisms by which the vacuolar proteins reach their destinations. As with other aspects of the secretory system, some of the findings will be applicable to all eukaryotic cells, while others will be specific for plant cells. ***