Dimer formation of subunit G of the yeast V‐ATPase

FEBS Letters

Godovac-Zimmermann, Jasminka⁴  Koch, Michel H.J³  Grüber, Gerhard¹  Bailer, Susanne M²  Armbrüster, Andrea¹ 

[1] Universität des Saarlandes, Fachrichtung 2.5 – Biophysik, D-66421 Homburg, Germany;Universität des Saarlandes, Fachrichtung 2.3 – Medizinische Biochemie und Molekularbiologie, D-66421 Homburg, Germany;European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, D-22603 Hamburg, Germany;University College London, Centre for Molecular Medicine, London WC1E 6JJ, UK

关键词: Vacuolar-type ATPase;    V1VO ATPase;    V1 ATPase;    Vma10p;    Dimerization;    Small-angle X-ray scattering;    BSA;    bovine serum albumin;    IPTG;    isopropyl (thio)-β-D-galactoside;    NTA;    nitrilotriacetic acid;    PAGE;    polyacrylamide gel electrophoresis;    PCR;    polymerase chain reaction;    SDS;    sodium dodecyl sulfate;    Tris;    Tris-(hydroxymethyl)aminomethane;   

DOI  :  10.1016/S0014-5793(03)00643-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V₁ and V_O sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28±2 kDa, indicating that this protein is dimeric. With a radius of gyration (R _g) and a maximum size (D _max) of 2.7±0.2 nm and 8.0±0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G_38–144 form (the carboxyl-terminus) was expressed and purified. G_38–144 is homogeneous, with a molecular mass of approximately 12±3 kDa, indicating a monomeric form in solution.

【 授权许可】

Unknown