The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and gold nanoparticle technique

利用原子力顯微鏡和金奈米粒子技術解析雙元體液泡質子傳送焦磷酸水解酶之羧酸端間距

劉振凰(Tseng-Huang Liu)

指導教授 ： 潘榮隆 ； 曾繁根

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2010年)

摘要

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen at the expense of PPi hydrolysis for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 ± 0.3 and 26.3 ± 4.7 nm, whereas those of the luminal protrusions were 1.2 ± 0.1 and 21.7 ± 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of membrane and close to each other as visualized with antibody and gold nanoparticles against 6His tag on C-ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 4.2 ± 1.4 nm. Thus our study is the first to provide the structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.

關鍵字

焦磷酸水解酶 ； 原子力顯微鏡 ； 奈米金粒子 ； 電子顯微鏡

參考文獻

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