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

Abstract

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 α-helical segments of the G and E subunits are closely aligned over a distance of up to 40 Å. 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 ∼ 150 Å long parallel α-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.

Keywords::

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

Acknowledgements

We thank Ms Meital Cohen for assisting with cross-linking experiments.

Declaration of interest: This work was supported by the UK BBSRC (grant numbers 24/b14097 and BB/D016142/1). The MassPREP and MALDI instruments were funded through a BBSRC JREI (2000) award. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.