標題: The proximity between C-termini of dimeric vacuolar H(+)-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique
作者: Liu, Tseng-Huang
Hsu, Shen-Hsing
Huang, Yun-Tzu
Lin, Shih-Ming
Huang, Tsu-Wei
Chuang, Tzu-Han
Fan, Shih-Kang
Fu, Chien-Chung
Tseng, Fan-Gang
Pan, Rong-Long
材料科學與工程學系奈米科技碩博班
Graduate Program of Nanotechnology , Department of Materials Science and Engineering
公開日期: 1-八月-2009
摘要: Vacuolar H(+)-translocating inorganic pyrophosphatase [vacuolar H(+)-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 PP(i) 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 nm and 26.3 +/- 4.7 nm, whereas those of the luminal protrusions were 1.2 +/- 0.1 nm and 21.7 +/- 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6xHis tags on C-terminal ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 2.2 +/- 1.4 nm. Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.
URI: http://dx.doi.org/10.1111/j.1742-4658.2009.07146.x
http://hdl.handle.net/11536/6875
ISSN: 1742-464X
DOI: 10.1111/j.1742-4658.2009.07146.x
期刊: FEBS JOURNAL
Volume: 276
Issue: 16
起始頁: 4381
結束頁: 4394
顯示於類別:期刊論文