INDUCTION OF AN ORGANELLE: THE YEAST PEROXISOME

过氧化物体是一种重要的细胞器，在几乎所有 真核细胞。它们催化各种氧化、代谢 相互转化和生物合成反应。过氧化物酶缺陷症 人类的集合体是儿童灾难性疾病的罪魁祸首 Zellweger综合征和新生儿肾上腺脑白质营养不良 很小的时候。虽然现在已经知道了很多关于 过氧酶体，人们对它们的组装了解得很少 细胞器。这项研究的长期目标是理解 靶向膜的分子相互作用 过氧化体蛋白的移位或组装和成熟。 将有两种酵母菌：波氏假丝酵母和酿酒酵母 利用他们的长处来描述过氧化物体的三个方面 集合。(1)过氧化体整体膜上的靶向序列 蛋白质PMP47将被鉴定出来。这种蛋白质的融合来自 甲醇酵母C boidinii和二氢叶酸还原酶 建造的。它们在体内分选到S。 酿酒酵母将通过细胞分离技术和 免疫印迹和免疫显微镜。PMP47部分中的嵌套删除 的融合将被生成以识别假定的靶向序列。 这样的序列将在不同的结构中得到确认。允许 将确定目标信号(S)的变化。抗体 抗PMP47和二氢叶酸还原酶将在这些研究中使用。 (2)BP48的功能，这是一种特异性和竞争性的蛋白质 在体外结合到过氧化体靶向序列，将被分析。BP48 将通过亲和层析纯化，并将提高抗体 反对它。这些将被用来定位细胞内的蛋白质。一个 脉冲追逐和免疫共沉淀方案，使用BP48抗体 以确定是否与新合成的过氧化物体结合 蛋白质存在于体内。相应的基因将被分离出来，并且一个 将在酿酒酵母中构建破坏性菌株，以进一步评估 该蛋白在过氧化体组装中的重要性。(3)职能 在酿酒酵母中丰富的三种过氧化物体膜蛋白中， 将证明scPMP24、scPMP31和scPMP32。尚未执行任何函数 显示为任何特定的真正的过氧体完整膜蛋白。 编码这些蛋白质的基因将利用多肽进行克隆。 来自纯化蛋白质的序列或通过与相似的蛋白质的同源性 波氏拟线虫过氧化体膜的大小。的功能的线索 可以从推断的一级结构中获得scPMP。菌株 包含被破坏的基因，如果可行，将由各种研究人员进行研究 用酶分析、形态分析和生物合成分析来评估 这些蛋白质对过氧化体的组装和功能起作用。如果合适的话， 基因将被放置在受调控的启动子上，以研究不同 对功能的表达水平。如果没有观察到表型，菌株 包含中断的组合，将生成以测试 冗余功能。最终，函数的假设将在 重组的系统。这些基因也将对未来的研究有用。 这类蛋白质的组装和拓扑结构。

Peroxisomes are essential organelles that are found in virtually all eukaryotic cells. They catalyze a variety of oxidations, metabolic interconversions and biosynthetic reactions. Defects in peroxisomal assembly in humans are the coarse of the catastrophic childhood diseases Zellweger syndrome and neonatal adrenoleukodystrophy which lead to death at an early age. Although much is now known about the functions of peroxisomes, considerably less is understood about the assembly of these organelles. The long-term goal of this research is the understanding of the molecular interactions responsible for the targeting, membrane translocation or assembly, and maturation of peroxisomal proteins. Two yeast species Candida boidinii and Saccharomyces cerevisiae will be utilized for their strengths in characterizing three aspects of peroxisomal assembly. (1) Targeting sequences on a peroxisomal integral membrane protein, PMP47, will be identified. Fusions of this protein from the methylotrophic yeast C boidinii, and dihydrofolate reductase will be constructed. Their sorting in vivo into the peroxisomal membrane of S. cerevisiae will be monitored by techniques of cell fractionation and immunoblotting, and immunomicroscopy. Nested deletions in the PMP47 part of the fusions will ge generated to identify putative targeting sequences. Such sequences will be confirmed in separate constructions. Allowed alterations of the targeting signal(s) will be determined. Antibodies against PMP47 and dihydrofolate reductase will be used in these studies. (2) The function of BP48, a protein that specifically and competitively binds to a peroxisomal targeting sequence in vitro, will be analyzed. BP48 will be purified by affinity chromatography and antibodies will be raised against it. These will be used to localize the protein within the cell. A pulse-chase and co-immunoprecipitation protocol, using the BP48 antibodies will be performed to determine if binding to newly synthesized peroxisomal proteins occurs in vivo. The corresponding gene will be isolated and a disruption strain in S. cerevisiae will be constructed to further assess the importance of this protein in peroxisomal assembly. (3) The function of the three abundant peroxisomal membrane proteins in S. cerevisiae, scPMP24, scPMP31, and scPMP32, will be proved. No functions have yet been shown for any specific bona fide peroxisomal integral membrane protein. The genes encoding these proteins will be cloned wither using peptide sequences from the purified proteins or by homology to proteins of similar size in peroxisomal membranes of C. Boidinii. Clues to function of the scPMPs may be obtained from the inferred primary structures. Strains containing the disrupted genes, if viable, will be studied by a variety of enzymatic, morphological, and biosynthetic assays to assess the role of these proteins on peroxisomal assembly and function. If appropriate, the genes will be placed on a regulated promoter to study effects of different levels of expression on function. If no phenotype is observed, strains containing a combination of disruptions will be generated to test for redundant functions. Ultimately, hypotheses of function will be tested in reconstituted systems. The genes will also be useful for future studies of the assembly and topology of this class of proteins.