Page 177 - The Biochemistry of Inorganic Polyphosphates

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WU095-08
WU095/Kulaev
Yeast 161
The syringomycin caused P i efflux from the cells of Rhodotorula pillimanae, with the
efflux being accompanied by a decrease in the PolyP content and acidification of the
cytoplasm (Reidl et al., 1989). 31 P NMR spectroscopy performed on xylose-grown whole
cells of Candida tropicalis showed that azide lowered the intracellular pH, inhibited the P i
uptake, and decreased the building of PolyP (Lohmeier-Vogel et al., 1989). A similar re-
sult was obtained with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP)
(Lohmeier-Vogel et al., 1989).
It was shown that the P i uptake and PolyP accumulation in S. cerevisiae is suppressed by
antimycin A if ethanol is used as an energy source and is not suppressed in the presence of
glucose (Schuddemat et al., 1989a). This fact indicates that the miotochondrial function may
be important for PolyP accumulation when oxidative phosphorylation is the main energy
source.
The effect of CCCP on ‘NMR-detected’ PolyP was investigated in S. cerevisiae grown
on lactate (Beauvoit et al., 1991). The cells were incubated in a resting medium in aerobiosis
with lactate or glucose or in anaerobiosis with glucose. For each case, in vivo 31 P NMR
spectroscopy was used to measure the levels of phosphorylated compounds. A spontaneous
PolyP breakdown occurred in anaerobiosis and in the absence of CCCP. In aerobiosis,
PolyP hydrolysis was induced by the addition of either CCCP or a vacuolar membrane
ATPase-specific inhibitor, bafilomycin A1 (Beauvoit et al., 1991).
It is important to note that selection of the concentrations of the uncouplers, which are
inhibitory for PolyP accumulation but not for P i uptake, is a difficult task. In many cases, the
decrease of PolyP content in the presence of membrane-damaging agents and ionophores
may be due to a break in P i uptake and a lack of P i for PolyP synthesis. The latter may
decrease independent of the form of P i -uptake inhibition. For example, Candida humicola
accumulated 10-fold more PolyP during active growth in a complete glucose–mineral salt
medium, pH 5.5, than at pH 7.5. This is probably due to the high P i uptake rate from the
culture medium at pH 5.5, whereas a 4.5-fold decrease in P i uptake occurred at pH 7.5
(McGrath and Quinn, 2000).
Despite the above circumstances, we have attempted to analyse the effects of some in-
hibitors on PolyP accumulation in glucose-grown S. cerevisiae during phosphate overplus
(Trilisenko et al., 2003). The protonophore FCCP suppressed PolyP accumulation, indicat-
ing the dependence of this process on the µH on the membranes (see Figure 8.24). The
+
PolyP(IV) fraction was shown to be the most sensitive, with the PolyP(I) fraction the next
in sensitivity to FCCP (Figure 8.24). Iodoacetamide, a well-known inhibitor of glycoly-
sis, had little effect despite the effective growth inhibition. In the presence of bafilomycin
A1, the increases in the contents of PolyP(I), PolyP(IV) and PolyP(V) during phosphate
overplus were the same as in the control, whereas the contents of PolyP(II) and PolyP(III)
were lower than in the control. The synthesis of some parts of these fractions probably
+
depends on the µH on the vacuolar membranes. In the presence of cycloheximide, an
inhibitor of protein synthesis, only the accumulation of the PolyP(IV) fraction appeared to
be disrupted. The insignificant effect of cycloheximide on the accumulation of other PolyP
fractions indicates that the corresponding enzymatic system was already induced during
phosphorus limitation. The unequal effects of the inhibitors on the accumulation of certain
PolyP fractions confirms the idea that these fractions have specific pathways of biosynthesis
and a specific function in yeast cells.