N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

Abstract

V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

The N-terminal domain of the V-ATPase subunit 'a' is regulated by pH in vitro and in vivo

“…20 Similar to our study, Merkulova and co-workers 20 found that the mammalian a2 subunit consists of mostly alpha helical fold and elutes in multiple peaks from a gel-filtration column, consistent with oligomerization of this domain. Strikingly, the protein was stabilized at high pH, indicating similar pH-dependent properties.…”

“…1D), which was consistent with previous observations and secondary structure predictions (ref. 20 and data not shown).…”

Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase

“…1B). Earlier studies have shown that constructs including more or all the N-terminal residues proved difficult to work with because of the tendency of the proteins to aggregate and susceptibility to degradation (37,38). We therefore decided to generate N-and C-terminal truncations to identify minimal soluble and folded domains that could serve in subunit-subunit interaction studies.…”

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

“…The positions of the A (yellow), B (red), C (brown), E (purple), G (beige), H (orange), and DF (blue) subunits and c-ring (magenta) are also shown (5). The N-terminal cytosolic tail of V-ATPase a-subunit (aN) is situated in parallel to the membrane plane and could have contacts with membrane (66). The position of first 17-amino acid peptide-forming epitope (aN(1-17)) involved in interaction with the Sec-7 domain of cytohesin-2 is also indicated.…”