Crystal structure of yeast V1-ATPase in the autoinhibited state (2016)
Figure 5

Figure 5. Role of HCT in activity silencing

A Alignment of the amino acid sequence of the yeast H subunit inhibitory loop region (shown in Fig 4C). The yeast (Sc) loop was aligned with those from insect (M. sexta), zebrafish (D. rerio), frog (X. trop), mouse (Mus), bovine (B. taur), chimpanzee (P. trog) and human (Hum1, Hum2), illustrating the lack of sequence conservation in this region. Pink coloring denotes predicted α helical secondary structure.

B H subunit mutations. Left panel, schematic representation of the H subunit mutations analyzed. Note that D410 is the only conserved residue in the inhibitory HCT loop. Right panel, overlay of the inhibitory HCT loops from yeast H (yellow) with homology models of the human H (HsH, purple) and yeast H subunit mutant HLoop (cyan).

C Growth phenotype of H subunit mutants. Deletion of V-ATPase subunits from yeast results in a conditional lethal phenotype (Vma-) characterized by an inability to grow at pH 7.5 in the presence of 60 mM CaCl2 (upper panel), but growth is observed at pH 5 (lower panel). Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed. Both human H subunit isoforms (HsH1, HsH2), a chimeric H subunit (HChim) containing ScHNT and HsHCT, mutations in yeast intended to mimic the length of the human H loop sequence and accessibility (Hloop), and a point mutation of the conserved aspartic acid residue in the loop sequence (HD410A) are shown compared to the H subunit deletion (ΔH) and wild type H on a plasmid (H WTpl). All grow on pH 5 medium, but the human isoforms failed to complement the deletion phenotype.

D ATPase activity of H subunit mutants. The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH, for comparison of MgATPase activity. Average specific activities are shown plotted +/- s.e.m from two independent purifications. The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity (Parra et al, 2000; Zhang et al, 2003), whereas ScV1 containing plasmid-borne wild type H (H WTpl) exhibits low levels of activity likely due to reduced expression from the low copy plasmid. The HChim and HLoop mutants lead to a loss of activity silencing, displaying nearly the same activity as ScV1ΔH.

E SDS-PAGE and Western Blot analysis of ScV1 preparations. Left panel, 15% SDS PAGE of ScV1 purified from the subunit H mutant and WT strains. Note that H WTch is purified from a yeast strain deleted for the C subunit (used for crystallization). The remaining strains contain subunit C, which is co-purified with ScV1 in varying levels. Note that while H WTch ScV1, which is expressed from a strain deleted for subunit C, displays no detectable MgATPase activity, the H subunit mutants co-purified with the most C subunit display the least activity, suggesting that absence or presence of subunit C has no effect on MgATPase activity. Right panel, immunoblot probed with an antibody directed against the N-terminal Myc tagged mutant H subunits expressed from a plasmid. Note that the levels of mutant H expression does not appear to vary from strain to strain.