Vacuolar Protein Sorting in Plant Cells

9315940 Chrispeels，Marten J. 该提案的目标是了解蛋白质如何靶向并积累在植物细胞的液泡中。 虽然植物液泡与酵母液泡和哺乳动物细胞的溶酶体有关，但它们含有更多种类的蛋白质，包括贮藏蛋白、凝集素、酶抑制剂、防御酶以及许多常见于所有液泡和溶酶体中的酸性水解酶。 在过去的几年里，在理解液泡蛋白本身上发生的分选信号方面已经取得了很大进展，但是目前还没有关于允许这些蛋白正确靶向其目的地的细胞机制的信息。 高尔基体衍生的囊泡的正确液泡靶向涉及囊泡外部的蛋白质，其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到膜上的蛋白质包被和去包被，并且可以从胞质溶胶中募集。鉴定这些蛋白质是本提案的主要目标。 Pi将利用最近分离的编码拟南芥磷脂酰肌醇3-激酶的基因，并研究它是否在液泡蛋白分选中发挥作用。 该蛋白的功能将在稳定转化和瞬时系统或用反义寡脱氧核糖核苷酸处理的原生质体中进行检查。 Pi将用植物磷脂酰肌醇3-激酶基因转化酵母vps 34突变体以寻找相互作用的蛋白质，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还将继续致力于将蛋白质靶向液泡膜或液泡膜。 他将构建一种报告蛋白，并针对该蛋白制备抗血清，以及不同的跨膜结构域。 这项工作的意义在于更好地理解植物细胞中液泡的生物发生。 本文的研究将有助于我们理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，而另一些则是植物细胞特有的。 ***

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. ***