Peroxisome Biogenesis in the Yeasts Candida tropicalis and Saccharomyces cerevisiae

过氧化物体在真核细胞中几乎无处不在，在呼吸和脂类代谢中具有重要功能。过氧体蛋白是核编码的，在游离的多聚核糖体上合成，然后在翻译后输入到预先存在的过氧体中。与许多线粒体和叶绿体蛋白不同，过氧化体蛋白是在成熟大小的时候合成的，缺乏可切割的转运肽。这个项目的长期目标是了解过氧化酶体组装的机制。这项建议的目的是鉴定将过氧化物体蛋白定向到酵母过氧化物体的拓扑致病序列。在此之前，该实验室确定了从热带假丝酵母中体外进口过氧化物体蛋白的条件，并在一种过氧体蛋白(乙酰-辅酶A氧化酶)中鉴定了能够将该蛋白定向到过氧酶体的拓扑形成序列。关于靶向和插入过氧化物体整合膜蛋白的情况尚不清楚。该计划是从热带毛囊线虫过氧化体中克隆和测序一个基质和膜蛋白。使用建立的体外试验和体内进口试验，将调查和比较过氧化物体靶向信号。另外，来自酿酒酵母的过氧化物体柠檬酸合成酶基因已经在手中；这将被表达，并将使用上述方法，将确定信号序列是否在来自热带酵母和酿酒酵母的过氧化体蛋白中保守。通过这些手段，人们希望能更好地了解过氧化物酶体内组装的机制。

Peroxisomes are nearly ubiquitous in eukaryotic cells and have essential functions in respiration and lipid metabolism. Peroxisomal proteins are nuclear-encoded and are synthesized on free polyribosomes, then imported into pre-existing peroxisomes post-translationally. Peroxisomal proteins, unlike many mitochondrial and chloroplast proteins, are synthesized at their mature size and lack cleavable transit peptides. The long term objective of this project is to understand the mechanism of peroxisome assembly. The aims of this proposal are to identify topogenic sequences that act to direct peroxisomal proteins to yeast peroxisomes. Previously, this laboratory determined conditions for measuring the in vitro import of peroxisomal proteins to peroxisomes from the yeast Candida tropicalis, and identified topogenic sequences in one peroxosomal protein (aceyl-CoA oxidase) that are capable of direction this protein to peroxisomes. Nothing is known regarding the targeting and insertion of peroxisomal integral membrane proteins. The plan is to clone and sequence a matrix and membrane protein from C. tropicalis peroxisomes. Using the established in vitro assay and by an in vivo import assay, peroxisomal targetting signals will be investigated and compared. Also, the gene encloding peroxisomal citrate synthase from Saccharomyces cerevisiae is in hand; this will be expressed and, using the above methods, it will be determined whether signal sequences are conserved in peroxisomal proteins from C. tropicalis and S. cerevisiae. By these means, it is hoped to gain a better understanding of the mechanism by which peroxisomes are assembled.