酵母菌過氧化體生合成的必要蛋白Pex19p的結構與功能關係探討

The structural and functional studies of Pex19p required for yeast peroxisome biogenesis

林育緯(Yu-Wi Lin)

指導教授 ： 蔡榮宗

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2010年)

https://doi.org/10.6834/CSMU.2010.00044

摘要

過氧化體合成相關的蛋白稱為peroxin，其編碼基因以PEX命名，大部分過氧化體蛋白在細胞質中製造，再經過特殊的運輸機制將過氧化體的蛋白送至過氧化體，最後形成功能完整的過氧化體，其中以Pex19p對於過氧化體的生合成的功能最為重要，Pex19p主要具有伴侶蛋白和過氧化體膜蛋白受體的作用，大部分的過氧化體膜蛋白都是藉由Pex19p運輸至過氧化體膜上。而Pex19p在人類部分被認為是以單體型態存在，但實際大小卻比預估更大，推測是N端結構鬆散關係，在植物的阿拉伯芥上Pex19p則是以二聚體的型態存在，酵母菌中有研究指出Pex19p是單體型態，突變後卻變的更大，這種型態上的不穩定情形令我們想要探討酵母菌Pex19p是否存在除了單體以外更大的複合體。我們製備了帶有His Tag的Pex19p全長、Pex19p蛋白C端和N端的重組蛋白，經鎳離子親合性管柱純化後，使用膠體過濾層析法來鑑定Pex19p全長、N端、C端重組蛋白的構型，經分子大小換算後，發現His-Pex19p重組蛋白可能會形成四聚體，並且發現只包含Pex19p蛋白C端或N端的重組蛋白，其複合體的穩定性較差而呈現兩種分子型態。我們更進一步利用油酸誘導Pxa1p來分析Pex19p的結構與功能相關性，另外，我們也使用酵母菌雙雜交法來分析Pex19p本身相互作用的區域，此外我們也發現Pex19p在大腸桿菌中具有使刪除穿膜區的Pxa1p(561-870)蛋白含量增加的能力

關鍵字

過氧化體 ； 過氧化體生合成

並列摘要

Peroxisome synthesis-related proteins are called peroxins and encoded by PEX genes. Most of peroxins are translated in the cytoplasm, then sent to peroxisome through a special transport mechanism and finally form a fully functional peroxisome. Among these peroxins, Pex19p is the most important one in peroxisome biogenesis. It has been proposed that Pex19p has chaperone-like and receptor functions for peroxisome membrane protein ( PMP ), because it bind and stabilize newly synthesized PMPs in the cytoplasm. Most of the peroxisome membrane proteins are transported to peroxisome in Pex19p-dependent manner. Human Pex19p are considered as a monomer, but its actual size was larger than expected. This property has been attributed to the flexibility of N-terminal half of Pex19p. In the Arabidopsis, PEX19 are proved to form both monomer and dimer, and the dimer form was suggested to be the functional form. In Saccharomyces cerevisiae, Pex19p has been determined as a monomer. When mutated in C-terminal farnesylation site of yeast Pex19p, its molecular weight has become larger than expected. We propose that the significant change of molecular weight of Pex19p caused by mutation may indicate that Saccharomyces cerevisiae Pex19p has the molecular forms other than monomer. So, We prepared the purified full-length, N-terminal and C-terminal recombinant Pex19p proteins with a N-terminal His-tag by nickel ion affinity column and used gel filtration chromatography to identify the molecular weights of these protein. The molecular weights of full-length and mutant Pex19p proteins were determined by comparing the retention time of these proteins with the retention time of molecular weight standards. It turns out that full-length Pex19p may form a tetramer, but N-terminal or C-terminal half combinant Pex19p proteins were less stable and showed two molecular forms with more or less numbers than full-length one. We further establish the functional assay of Pex19p to analyze its structural and functional correlation by using Pxa1p total protein contents as an indicator. We also used the yeast two-hybrid method to analyze the Pex19p interaction region with itself. We also found that Pex19p has the ability to increase total protein contents of the N-terminal transmembrane region deleted Pxa1p561-870 in Ecoli,indicating an Pex19p-interacting motif existed in the c-terminal fragment of Pxa1p.

並列關鍵字

peroxisome ； Pex19p ； peroxisome biogenesis ； Gel filtration

參考文獻

1. de Duve C, Baudhuin P. (1966). Physiol.Rev.46, 323–57

7. Subramani, S. (1997). Nat. Genet. 15, 331–333.

9. Fujiki, Y. (1997) Biochim. Biophys. Acta 1361, 235–250.

12. Dodt,G. and Gould,S.J. (1996) J. Cell Biol 135, 1763–1774.

13. Fransen,M., Terlecky,S.R. and Subramani,S. (1998). Proc. Natl Acad. Sci. USA, 95, 8087–8092.