New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis - PubMed (original) (raw)
New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis
N Ogawa et al. Mol Biol Cell. 2000 Dec.
Free PMC article
Abstract
The PHO regulatory pathway is involved in the acquisition of phosphate (P(i)) in the yeast Saccharomyces cerevisiae. When extracellular P(i) concentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor. In an attempt to identify all the components regulated by this system, a whole-genome DNA microarray analysis was employed, and 22 PHO-regulated genes were identified. The promoter regions of 21 of these genes contained at least one copy of a sequence that matched the Pho4 recognition site. Eight of these genes, PHM1-PHM8, had no previously defined function in phosphate metabolism. The amino acid sequences of PHM1 (YFL004w), PHM2 (YPL019c), PHM3 (YJL012c), and PHM4 (YER072w) are 32-56% identical. The phm3 and phm4 single mutants and the phm1 phm2 double mutant were each severely deficient in accumulation of inorganic polyphosphate (polyP) and P(i). The phenotype of the phm5 mutant suggests that PHM5 (YDR452w) is essential for normal catabolism of polyP in the yeast vacuole. Taken together, the results reveal important new features of a genetic system that plays a critical role in P(i) acquisition and polyP metabolism in yeast.
Figures
Figure 1
PHO regulation system. A simplified schematic of the PHO regulation mechanism showing the five main regulator proteins is illustrated (Johnston and Carlson, 1992; Oshima, 1997). Ovals and boxes represent proteins and genes, respectively. Thick lines mean that the signals are transduced to the downstream component, while dotted lines indicate the absent of an interaction with the downstream component. Open ovals and boxes indicate active states, gray oval and boxes indicate inactive state. The _PHO5_-constitutive mutants used in this study are listed to the right of the corresponding mutated protein with the nature of the mutation indicated by “gain” or “loss” of function.
Figure 2
DNA microarray analysis. (A) Fluorescent scanning images of the spots for the typical PHO-regulated genes,PHO5, PHO84, and_YPL019c/PHM2_, and a non PHO-regulated gene,CDC19. The column number indicates experiments performed as follows, 1 and 2: comparison between NBW7 (wild-type) cultivated in low- (YPAD-Pi, Cy5) and high-Pi media (YPAD+Pi, Cy3); 3: DBY7286 (wild-type) cultivated in low- (YPAD-Pi, Cy5) and high-Pi media (YPAD+Pi, Cy3); 4: comparison between NBD82–1 (PHO4_c_-1, Cy5) and NBW7 (Cy3); 5: comparison between NBD80–1 (_pho80_Δ, Cy5) and NBW7 (Cy3); 6: comparison between NBD85A-1 (_pho85_Δ, Cy5) and NBW7 (Cy3); 7 and 8: comparison between NOF1 (PHO81_c_-1, Cy5) and NBW7 (Cy3). All strains used in experiment 4, 5, 6, 7, and 8 were cultivated in YPAD media. (B) Cluster analysis of the DNA microarray data. Eight independent DNA microarray data were analyzed the Cluster program (Eisen et al., 1998). Genes showing more than twofold induction in any experiments correspond to the rows, and the experiments are the columns. Red represents a higher level of expression in the low-Pi conditioned wild-type cells compared with the high-Pi conditioned cells or in the mutant strains compared with the wild-type strains. The color saturation represents the magnitude of the expression ratio, as indicated by the scale at the top left of the figure. Black indicates no detectable difference in expression levels; gray denotes a missing observation. The rows, representing series of observation on individual genes, were ordered based on the similarity of their expression patterns in the eight experiments (Eisen et al., 1998). The dendrogram to the left of the figure represents the correlation between expression patterns, as described by Eisen et al., (1998). Names of the genes listed in Table 2 are emphasized by red color.
Figure 3
(A) Homology between Phm1, Phm2, Phm3, and Phm4. Shaded and white boxes represent homologous and nonhomologous regions, respectively, and their identities to the Phm1 sequence are indicated by percentage. The region homologous to Pho81 and the putative transmemembrane region are indicated above the Phm1 box. (B) Consensus sequences in the N-terminal regions of nine S. cerevisiae proteins, including Pho81. Hydrophobic, positive- and negative-charged amino acid residues are indicated by “*,” “+,” and “-,” respectively. (C) The putative Phm5 protein structure. The region of sequence similarity to acid sphingomyelinase in human and C. elegans is indicated by a shaded box. The putative transmembrane region is indicated by a filled box.
Figure 4
PolyP analysis by PAGE for various_phm_ mutants. Each polyP sample, containing 10-μg RNA, prepared from indicated strains after the polyP overplus culture, was loaded on 20% polyacrylamide gel, and subjected to electrophoresis at 20 V/cm for 1.5 h (panel A) or 3 h (panel B). The strains used were NBW7 (WT), NBM4Lf1 (_phm1_Δ), NBM19Hf1 (_phm2_Δ), NBM4L19H2 (_phm1_Δ_phm2_Δ), NBM12W2 (_phm3_Δ), NBM72W7 (_phm4_Δ), NBM452H1 (_phm5_Δ), NBM18H7 (_ctf19_Δ), NBM281H1 (_phm6_Δ), NBD4–1 (_pho4_Δ), NBD80–1 (_pho80_Δ), and NBD8184–1 (_pho84_Δ). Several RNA bands are visible in a region near the top of the both gels, indicated by “RNAs”. Marker samples containing 10 μg of sodium phosphate glass type 5, 15, and 35 were loaded on the P5, P15, and P35 lanes, respectively. Extracted polyP samples corresponding to 55 and 65 Pi residues were also loaded as size markers (PP55 +65). Chain lengths of the polyP ladders determined by the markers (Materials and Methods) are indicated to the left and right of panel A and B, respectively. The migration positions indicated with the chain length in the parenthesis on panel B are estimated from a standard curve.
Figure 5
Localization of Phm2-GFP. The identical frames of the yeast transformant NBM4L19H1[pPHM2-GFP] were shown for Normarsky (left column), FM 4–64 fluorescence(center column), and GFP fluorescence (right column) images.
Figure 6
(A) PolyP PAGE analysis of_vma4_ and pep4 mutants. PolyP samples were prepared from the strains, CRY-V4 (_vma4_Δ), CRY (WT), CRX (_ppx1_Δ), CB024 (Pro), and NB91–6A (pep4) cultivated by the polyP overplus method. The other conditions were as described in Figure 4A. (B) PolyP PAGE analysis of vma4 and phm mutants cultivated at low pH. PolyP samples were prepared from the strains, CRY (WT), CRY-V4 (_vma4_Δ), NBM4L19H2 (_phm1_Δ _phm2_Δ), NBM12W2 (_phm3_Δ), and NBM72W7 (_phm4_Δ) cultivated in YPAD-Pi media, followed by cultivation in YPAD supplemented with either 10 mM Pi (Pi), or 10 mM Pi and 10 mM sodium acetate buffer (Ac), for 2 h. The other conditions were as described in Figure 4A.
Figure 7
Ca2+ sensitivities of_phm_ mutants at neutral pH. CRY (WT-1), CRY-vma4 (_vma4_Δ), NBW7 (WT-2), NBM4L19H2 (_phm1_Δ _phm2_Δ), NBM12W2 (phm3Δ), and NBM72W7 (_phm4_Δ) were streaked on the YPAD plate (pH 7.5) supplemented with 60 mM CaCl2, and incubated at 30°C for 3 days.
Figure 8
Pi uptake in phm mutants. Pi uptake activities in the strains NBW7 (WT), NBM4Lf1 (_phm1_Δ), NBM19Hf1 (_phm2_Δ), NBM4L19H2 (_phm1_Δ _phm2_Δ), NBM12W2 (_phm3_Δ), NBM72W7 (_phm4_Δ), and NBD8184–1B (_pho84_Δ) were tested as described in Materials and Methods.
Figure 9
Pi acquisition and storage system in_S. cerevisiae_. The known of predicted roles of proteins encoded by the PHO-regulated genes (names indicated in red) are schematized. See text for details.
References
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- Bun-ya M, Shikata K, Nakade S, Yompakdee C, Harashima S, Oshima Y. Two new genes, PHO86 and PHO87, involved in inorganic phosphate uptake in Saccharomyces cerevisiae. Curr Genet. 1996;29:344–351. - PubMed
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