酵母菌過氧化體生合成因子PEX19的基因構築及其蛋白N端的 功能性探討

The gene construction of yeast peroxisome biogenesis factor 19 (PEX19) and the functional characterizations of the NH2-terminal region of Pex19p protein

葉美杏(Mei-Hsing Yeh)

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(2011年)

https://doi.org/10.6834/CSMU.2011.00161

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。而在Pex19p缺乏N-terminal domain D1的人類纖維母細胞會喪失過氧化體生合成功能，顯示Pex19p的N端區域在過氧化體生合成上扮演重要的角色，所以我們想要深入探討酵母菌Pex19的N端區域與過氧化體生合成的相關性。
先構築包含PEX19 promoter的質體，再以此質體為基礎，將全長Pex19p或Pex19p N端刪除之DNA序列接在Pex19p promoter的下游，再送入酵母菌中表現蛋白，全長Pex19p和Pex19p N端刪除之重組蛋白各自帶有HA蛋白標籤在其蛋白C端。接著我們利用油酸培養皿來進行功能性互補實驗，研究Pex19p N端刪除對過氧化體生成的影響。
然而，在實驗過程中我們發現，無HA蛋白標籤的全長Pex19p具有互補油酸使用的能力，但帶有HA蛋白標籤的全長Pex19-HA則無法互補成功，顯示C端接上HA蛋白標籤會影響Pex19p蛋白的功能。正因為如此，就無法判斷N端刪除對於Pex19p的功能有何影響，因為這些蛋白皆具有C端的HA蛋白標籤。因此，我們也進一步使用Western blot方式來分析全長Pex19p和Pex19p N端刪除突變蛋白在油酸培養基誘導下其蛋白的含量，作為功能分析的佐證。

關鍵字

過氧化體 ； 過氧化體生合成因子19

並列摘要

Peroxisome synthesis-related proteinsare called peroxins andencoded by PEX genes. Most of peroxinsare 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 haschaperone-like and receptor functions for peroxisome membrane protein ( PMP ), because it bind and stabilize newly synthesizedPMPs in the cytoplasm. Most of the peroxisome membrane proteinsare transported to peroxisome in Pex19p-dependent manner.Human fibriblasts will lose the function of peroxisome biogenesis if its Pex19p is lack of N-terminal domain D1；this phenomenon claims that Pex19p’s N-terminal region play a critical role in the process of peroxisome biogenesis. Hence，we would like to study the relatonship between N-terminal region and peroxisome biogensis.
First，construct the plasmid containing PEX19 promoter；Second，connect the full-length Pex19p’s DNA sequence with the downstram part of promoter；third，transfer it into yeast to express protein；fourth，repeat the same procedures on N-terminal deletion Pex19p. Full-length Pex19p and N-terminal deletion Pex19p both have HA-tag on their C-terminal. Sequently，we use oleic acid plate to proceed functional complementation test to study the effect upon peroxisome biogenesis of N-terminal region.
However，in the process of operating experiment，we found that full-length Pex19p without HA-tag has the function of oleic acid complementation and，in contrast，full-length Pex19p with HA-tag does not have the function of oleic acid complementation；therefore，we can not confirm the influence of N-terminal region due to the HA-tagt. Hence，we further use western blot to analyze quantities of protein that full-length Pex19p and N-terminal deletion Pex19p generates separately induced by oleic acid broth and take it as evidence of function alanysis.

並列關鍵字

peroxisome ； peroxisome biogenesis factor 19

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