INDUCTION OF AN ORGANELLE: THE YEAST PEROXISOME

蛋白质分类和蛋白质组装的基本机制 细胞器仍然是大部分未解决的。 特别是， 过氧化物酶体是一种重要的、普遍存在的细胞器，但对它的研究很少。 过氧化物酶体组装的缺陷可能导致灾难性的人类疾病。 甲醇诱导的酵母过氧化物酶体，结构和功能简单， 丰富和高度诱导，是一个很有前途的模型，研究基础 一般的细胞器生物发生和过氧化物酶体组装问题 特别的。 这项工作的长期目标是了解 过氧化物酶体蛋白分选信号与膜的相互作用 导致进口。 将采取两种方法来阐明组装 甲醇诱导的博伊丁假丝酵母过氧化物酶体。 (1)3-过氧化物酶体的结构、生物合成和组装途径 将研究膜蛋白。 工作将完成分析 新合成的PMP 20（过氧化物酶体膜蛋白20 kDa）的过氧化物酶体，并对编码该蛋白的基因进行测序。 这些研究将扩展到另外两种过氧化物酶体膜蛋白， 可能是PMP 31和PMP 47。 可用于以下的单克隆抗体： 免疫沉淀，现在可用于PMP 20，将提出对 其他膜蛋白用于此目的。 编码PMP 31和47的基因 将从现有的基因组文库中分离，并且它们的序列将 进行比较，以尝试识别可能对 将这些蛋白质定位于过氧化物酶体。 (2)分离的过氧化物酶体基因的体内表达系统 念珠菌将被开发，以便开始研究领域， 需要分类。 这些基因在大肠杆菌中的表达和分选 酿酒酵母将进行研究，以利用 这种生物体的遗传学知识和技术 可选择地， 将探索在博伊丁假丝酵母或另一种甲基营养菌中的表达。 这可能涉及营养缺陷型突变体和载体的发展， 转型 该系统将成为研究 基因改变（如缺失和融合）对定位的 改变蛋白质

The fundamental mechanisms of sorting of proteins and assembly of organelles are still largely unresolved. In particular, the biogenesis of peroxisomes, essential and ubiquitous organelles, has been little studied. Defects in peroxisomal assembly can lead to catastrophic human disease. The methanol-induced yeast peroxisome, simple in structure and function, abundant and highly inducible, is a promising model to study basic questions of organellar biogenesis in general and peroxisomal assembly in particular. The long range goal of this work is to understand the interactions of sorting signals on peroxisomal proteins with the membrane to cause import. Two approaches will be undertaken to elucidate assembly of the methanol-induced peroxisome in Candida boidinii. (1) The structure, biosynthesis, and assembly pathway of 3 peroxisomal membrane proteins will be studied. Work will be completed to analyze the pathway of newly synthesized PMP 20 (peroxisomal membrane protein of 20 kDa) to the peroxisome and to sequence the gene encoding this protein. These studies will be extended to two other peroxisomal membrane proteins, probably PMP 31 and PMP 47. Monoclonal antibodies that are useful for immunoprecipitation, now available for PMP 20, will be raised against the other membrane proteins for this purpose. The genes encoding PMP 31 and 47 will be isolated from an existing genomic library and their sequences will be compared to try to identify common domains which may be important for the targetting of these proteins to the peroxisomes. (2) A system for expression in vivo of the isolated peroxisomal genes from Candida will be developed, in order to begin to study domains which are necessary for sorting. Expression and sorting of these genes in Saccharomyces cerevisiae will be investigated in order to take advantage of the knowledge and techniques of genetics in this organism. Alternatively, expression in Candida boidinii or another methylotroph will be explored. This may involve the development of auxotrophic mutants and vectors for transformation. This system will be the basis for studying the effects of gene alterations (such as deletions and fusions) on localization of the altered proteins.