A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator

作者：Jones Richard P O; Durose Lyndsey J; Phillips Clair; Keen Jeffrey N; Findlay John B C; Harrison Michael A^*

来源：Molecular Membrane Biology, 2010, 27(4-6): 147-159.

DOI：10.3109/09687681003796441

• V-ATPase
• molecular motor
• Saccharomyces cerevisiae
• Enterococcus hirae
• vacuolar membrane
• cross-linking

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H+-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 angstrom. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two similar to 150 angstrom long parallel alpha-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.

• 出版日期2010-8