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

Tseng Huang Liu, Shen Hsing Hsu, Yun Tzu Huang, Shih-Ming Lin, Tsu Wei Huang, Tzu Han Chuang, Shih Kang Fan, Chien Chung Fu, Fan Gang Tseng, Rong Long Pan

研究成果: Article同行評審

12 引文 斯高帕斯(Scopus)

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 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 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 6×His 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.

原文English
頁(從 - 到)4381-4394
頁數14
期刊FEBS Journal
276
發行號16
DOIs
出版狀態Published - 2009 8月 1

All Science Journal Classification (ASJC) codes

  • 生物化學
  • 分子生物學
  • 細胞生物學

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