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Electron-microscopic structure of the V-ATPase from mung bean

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Abstract

The vacuolar H+-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V1 and Vo with overall dimensions of 25 nm×13.7 nm and a stalk region connecting the V1 and Vo domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.

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Fig. 1
Fig. 2a,b
Fig. 3a–d
Fig. 4a,b
Fig. 5

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Abbreviations

MSA :

Multivariate statistical analysis

2D, 3D :

Two-, three-dimensional

V-ATPase:

Vacuolar H+-ATPase

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Acknowledgments

We are very grateful to Prof. Fuyu Yang for continuous support and crucial advice on experiments. We thank G. Pfeifer and R. Hegerl for help with the EM program. This work was supported by grants from the Chinese Academy of Sciences (KSCX2-2-05) and the Third World Academy of Sciences (00-238 RG/Bio/AS) to Xujia Zhang. We thank Sarah Perrett and Judy Smith for their help in improving the manuscript.

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  1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China

    Zhuo Li & Xujia Zhang

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  1. Zhuo Li
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  2. Xujia Zhang
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Correspondence to Xujia Zhang.

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Li, Z., Zhang, X. Electron-microscopic structure of the V-ATPase from mung bean. Planta 219, 948–954 (2004). https://doi.org/10.1007/s00425-004-1298-2

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