The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique.
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.
Liu
Tseng-Huang
TH
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu, Taiwan, ROC.
Hsu
Shen-Hsing
SH
Huang
Yun-Tzu
YT
Lin
Shih-Ming
SM
Huang
Tsu-Wei
TW
Chuang
Tzu-Han
TH
Fan
Shih-Kang
SK
Fu
Chien-Chung
CC
Tseng
Fan-Gang
FG
Pan
Rong-Long
RL
D000906:Antibodies; D006046:Gold; D043564:Inorganic Pyrophosphatase / Q000737:chemistry*; D008051:Lipid Bilayers; D053768:Metal Nanoparticles; D018625:Microscopy, Atomic Force / Q000379:methods*; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D014617:Vacuoles / Q000201:enzymology
D000906:Antibodies; D008051:Lipid Bilayers; D010940:Plant Proteins; D006046:Gold; D043564:Inorganic Pyrophosphatase