以螢光顯微術研究蛋白質Pmp1p加入三元脂質人造膜的效應; The Effect of Protein Pmp1p on DPPC/POPC/Ergosterol Membranes

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题名:	以螢光顯微術研究蛋白質Pmp1p加入三元脂質人造膜的效應;The Effect of Protein Pmp1p on DPPC/POPC/Ergosterol Membranes
作者:	陳柏辰;Bo-Chen Chen
贡献者:	生物物理研究所
关键词:	氫離子幫浦;共軛焦顯微鏡;脂質浮排;人造膜;DPPC;POPC;Ergosterol;Pma1p;Pmp1p;Lipid raft;H+-ATPase;Model membrane;Confocal microscopy
日期:	2009-07-07
上传时间:	2009-09-22 10:21:47 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	Pmp1p是單次穿越細胞膜的小型蛋白質，主要的功能是調控細胞膜上的氫離子幫浦(H+-ATPase)，我們利用單層巨型微胞(GUV, giant unilamellar vesice)，其中膜成分包含了飽和脂質DPPC、不飽和脂質POPC、真核維生物固醇ergosterol和蛋白質Pmp1p，主要使用的儀器有共軛焦顯微鏡和螢光顯微鏡。我們改變溫度和Pmp1p的濃度去觀察單層巨型微胞(GUV, giant unilamellar vesice)的交互作用，並觀察單層巨型微胞(GUV, giant unilamellar vesice)人造膜的型態。添加Pmp1p到1:1:1 DPPC/POPC/ergosterol會影響到單層巨型微胞(GUV, giant unilamellar vesice)人造膜型態上的改變，當蛋白質濃度增加到0.2 mol% Pmp1p的過程中，亮區區塊(應為ld態)會隨之增加，暗區區塊(應為lo態)會隨之減少，這暗示了Pmp1p會使lo態的區塊比例減少，即脂質浮排(lipid raft)。另外Tmix的減少也指出了Pmp1p減少了有序的GUV人造膜。Pmp1p位在亮區區塊中，暗示Pmp1p較不喜歡位在脂質浮排(lipid raft)上，會優先地結合在非脂質浮排(non-lipid raft)。這與現行的觀點不同，由於Pmp1p能調控脂質浮排(lipid raft)上氫離子幫浦Pma1p的功能，因此推測兩者可能都在脂質浮排上。我們的結果指出Pmp1p和Pma1p是位在膜上不同區域，Pmp1p和Pma1p的調控過程是一種間接且尚未了解的機制。 Pmp1p is a small single-membrane span proteolipid that functions as a regulatory subunit of the plasma membrane H+-ATPase Pma1p. To explore the interaction of Pmp1p and lipids, we study the properties of GUV (giant unilamellar vesicle) model membranes composed of DPPC, POPC, ergosterol and protein Pmp1p by fluorescence and confocal microscopy. The fluorescence images of GUV membranes were taken as a function of temperature and Pmp1p concentration. The morphology of the GUV membranes is observed. Addition of Pmp1p to 1:1:1 DPPC/POPC/ergosterol influences the morphology of GUV membranes. The bright phase (presumably in the ld phase) increases, and the dark phase (presumably in the lo phase) decreases with increasing Pmp1p concentration up to 0.2 mol% Pmp1p. This suggests that Pmp1p decreases the fraction of the lo-phase regions, ie. lipid rafts. In addition, the decrease of Tmix also indicates that Pmp1p decreases the order of GUV membranes. It is found that Pmp1p locates in the bright-phase regions, implying that Pmp1p is preferentially associated with the non-raft region rather than the raft region. This is in disagreement with current views. It is believed that Pmp1p regulates the function of another raft protein H+-ATPase (Pma1p). Our results suggest that proteins Pmp1p and Pma1p exist in different parts of the membrane, and Pmp1p interacts with Pma1p indirectly via an unknown mechanism.
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