Page 98 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
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Enzymes of polyphosphate biosynthesis and degradation
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activity of high-molecular-weight exopolyphosphatase under phosphate overplus gradually
increased up to 10-fold in the logarithmic phase and then remained stable, while the activity
of the 40 kDa exopolyphosphatase decreased 1.3-fold in the same growth phase and dropped
almost 10-fold in the stationary growth phase. The high-molecular-weight exopolyphos-
phatase of cytosol was more active with PolyPs of longer chain lengths. Its activity with
PolyP 3 was only 13 % of that with PolyP 208 (Andreeva et al., 2001, 2004). The antibodies
against 40 kDa exopolyphosphatase had no effect on the high-molecular-weight ones. The
stimulation of activity by divalent cations was less in the case of high-molecular-weight
exopolyphosphatase when compared with the 40 kDa form (Table 6.4). High-molecular-
weight exopolyphosphatase of the cytosol also differed from exopolyphosphatases of the
cell envelope (Andreeva and Okorokov, 1993), nuclei (Lichko et al., 1996) and mitochon-
dria (Lichko et al., 1998), but was similar to the vacuolar exopolyphosphatase of the same
yeast (Andreeva et al., 1998b). It should be noted that, despite the simultaneousness of
PolyP accumulation and increase in high-molecular-weight exopolyphosphatase activity,
no direct interrelation has been found between these procecces. Cycloxemide blocked the
increase in high-molecular-weight exopolyphosphatase activity but had no effect on PolyP
accumulation.
The only known gene encoding the yeast exopolyphosphatase is the PPX1 gene, which
was cloned and sequenced (Wurst et al., 1995). The PPX1 gene belongs to the PPase C
family and has no sufficient similarity to the bacterial ppx gene (see http://www.expasy.org).
Exopolyphosphatase PPX1, a protein of 396 amino acids with a molecular mass of ∼ 45
kDa, was purified from a homogenate of S. cerevisiae (Wurst et al., 1995). A PPX1-deficient
strain was obtained using the gene elimination method (Wurst et al., 1995). Surprisingly, this
had an exopolyphosphatase activity of ∼ 50 % of the parent strain level. Thus, the existence
of other genes, encoding exopolyphosphatases in the yeast genome, was proposed (Wurst
et al., 1995).
Considerable changes in exopolyphosphatase spectrum were observed on PPX1 elim-
ination (Figure 6.6). In the PPX1-deficient strain, 40 kDa exopolyphospatases were not
observed in the cytosol, cell envelope and mitochondrial matrix (Lichko et al., 2002b,
2003a). Although PPX1 was absent in the cytosol of the mutant, exopolyphosphatase activ-
ity in this compartment decreased only twofold. This was explained by a concurrent fivefold
increase in the activity of high-molecular-weight exopolyphosphatase in this compartment,
whose properties were the same as those of the high-molecular-weight exopolyphosphatase
which appeared in the cytosol under phosphate overplus. No exopolyphosphatase activity
was found in a cell-envelope fraction of the PPX1 null mutant.
Inactivation of PPX1 did not result in any considerable changes in the content and
properties of vacuolar, nuclear and membrane-bound mitochondrial exopolyphosphatases
when compared with the parent strain of S. cerevisiae (Lichko et al., 2002b, 2003b). This
allows us to conclude that the 40 kDa exopolyphosphases of the cytosol, cell envelope and
the mitochondrial matrix of S. cerevisiae are encoded by the same PPX1 gene, and the
cytosolic high-molecular-weight enzyme and those of vacuoles, nuclei and mitochondrial
membranes are encoded by other genes (Lichko et al., 2002b, 2003a,b).
Distinction of the soluble mitochondrial exopolyphosphatase from those localized in the
yeast cytosol and cell envelope could be explained by post-translational modification of this
enzyme.
Under two different statuses of the yeast cell, P i overplus and PPX1 disruption, a
drastic increase in high-molecular-weight exopolyphosphatase activity and disappearance