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Characterization of the complex involved in regulating V-ATPase activity of the vacuolar and endosomal membrane

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Abstract

Regulator of the H+-ATPase of the vacuolar and endosomal membranes (RAVE) is essential for the reversible assembly of H+-ATPase. RAVE primarily consists of three subunits: Rav1p, Rav2p and Skp1p. To characterize these subunits, in this study, four strains derived from Saccharomyces cerevisiae BY4742 were constructed with a FLAG tag on the Rav1p and Rav2p subunits. Then, the corresponding RAVE containing complex was isolated by affinity purification. Western blot and MALDI-TOF mass spectrometry analyses showed that the RAVE complex contains not only the known V1-ATPase subunits (Vma1p and Vma2p) but also a newly found Leu1p that interacts with the RAVE subunit. Furthermore, we constructed rav1−/rav2−/vma2−/leu1-deficient recombinants by fusion PCR and homologous recombination and demonstrated that leu1 is indispensable in adjusting the microbial cell to adverse environments and that the function is similar to that of rav1/rav2 but significantly differs from that of vma2. Leu1p probably plays an important role in RAVE regulation of V-ATPase activity in conjunction with RAVE.

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Abbreviations

G418:

Geneticin

HRP:

Horseradish peroxidase

IgG:

Immunoglobulin G

PVDF:

Polyvinylidene difluoride

TBS:

Tris buffered saline

TBS/T:

TBS, 0.1% Tween-20

BSA:

Bovine serum albumin

DAB:

Diaminobenzidine

TFA:

Tallow fatty acid

ACN:

Acetonitrile

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Acknowledgements

The work was supported by the National Natural Science Foundation of China (30970058; 21176106) and Natural Science Foundation of Jiangsu Province (BK2012554), together with the Project Funded by China Postdoctoral Science Foundation (2015 M571666) and Fundamental Research Funds for the Central Universities (JUSRP51635B). Authors also would like to give the thanks to the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program.

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Zhenyu Zhang, Xiaojiao Wang, Tangjie Gao, Chunyin Gu, Fubao Sun & Lin Yu

  2. Faculty of Forestry, University of British Columbia, Vancouver, V6T 1Z4, Canada

    Jinguang Hu

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  1. Zhenyu Zhang
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  2. Xiaojiao Wang
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  3. Tangjie Gao
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  4. Chunyin Gu
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  5. Fubao Sun
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  6. Lin Yu
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Correspondence to Zhenyu Zhang or Fubao Sun.

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Zhang, Z., Wang, X., Gao, T. et al. Characterization of the complex involved in regulating V-ATPase activity of the vacuolar and endosomal membrane. J Bioenerg Biomembr 49, 347–355 (2017). https://doi.org/10.1007/s10863-017-9712-1

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  • DOI: https://doi.org/10.1007/s10863-017-9712-1

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