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Fig. 5. Schematic representation of the effect of bound antibody to Vma16p-HA on
V-ATPase activity. The V-ATPase is composed of two functional domains: the
catalytic V1 (A and B subunits) and the membranous V0
(a, c, c', c'' and d subunits), which
are interconnected by peripheral (C, E, G, H subunits) and central (D and F
subunits) stalks, respectively. It is assumed that hydrolysis of ATP by
V1 rotates the central shaft (D and F subunits), which in turn
causes the rotation of the membrane c-ring against its stator (E, G
and N'-a subunits), resulting in proton translocation into the
vesicle (Nelson et al., 2002 ;
Nishi and Forgac, 2002). It is
now clear that, similar to c and c' (Vma3p and Vma11p,
respectively), the c'' subunit (Vma16p) contains four TMs,
although in an opposite arrangement in the membrane with both N' and
C' pointing to the cytoplasm as shown above. The Vma16p (red) was tagged
with three HA-epitopes at its N terminus. When the resulting tagged V-ATPase
was incubated with anti-HA antibody, an inhibition of activity was observed.
This suggests that the binding of antibody (green) to the HA-tags interfered
with the rotation of the c-ring and shaft subunits (all presumed
rotating subunits are framed with a thick line). This interference is probably
due to a collision with the static subunits, and primarily with subunit
a, against which the rotor is turning. A similar effect was observed
when antibodies were allowed to bind to the Vma7p-HA (F subunit)-containing
complex. This is in contrast to the effect of antibody on the Vma10p-HA (G
subunit)-containing V-ATPase. Abs, bound anti-HA antibodies.
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yeast strain. (A)
Complementation of the yeast null mutant by the plant VMA16 cDNAs.
Yeast cells lacking the VMA16 gene were transformed with the pYES2
shuttle vector carrying the Arabidopsis thaliana (A-VMA16), lemon
fruit (L-VMA16) or the endogenous (Y-VMA16) cDNA. The growth phenotype of the
transformants was tested on YPD medium buffered at pH 5.5 or 7.5, on YPGal
plates, pH 7.5, with 2% galactose and 0.2% fructose, and minimal medium
without uracil (–URA). The yeast cells were grown for 4 days at
30°C, except for the YPGal plate that was left for 7 days at 30°C. WT,
wild type. (B) Protein sequence alignment of Vma16p from various species:
mouse (AF356006), human (D89052), C. elegans (Z68317), Citrus
lemon (AY226999) (this paper), Arabidopsis thaliana (AY226998)
(this paper) and S. cerevisiae (U_10399). Black boxes indicate
identical residues; shaded boxes indicate similar residues. Putative
transmembrane regions are indicated by dark lines labeled TM1–TM5.
HA). The ATP-dependent proton uptake activity was measured by
following the decrease in absorption difference at 490–540 nm of
Acridine Orange. Where indicated, 1 µmol of MgATP or 1 nmol of carbonyl
cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) was added to the
reaction mixture. WT, wild type.
2 µg protein) of the fraction with most
active ATPase (fraction no. 5 out of the 4–6 active fractions for
Vma16p-HA and Vma10p-HA; fraction no. 4 out of the 4–7 fractions for
Vma7p-HA; and fraction no. 7 out of the 5–8 active fractions for wild
type, WT), was preincubated in the presence and absence of 50 µl of 1:1000
(w/v) diluted anti-HA monoclonal antibody. 5 mmol l–1 MgATP
was added, and following 10 min incubation at 30°C, the reaction was
stopped and absorbance at 660 nm monitored. Each value represents a one single
experiment out of three.