Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism

Abstract

Background: Inorganic polyphosphate (poly P) occurs universally in all organisms from bacteria to man. It functions, for example, as a phosphate and energy store, and is involved in the activation and regulation of proteins. Despite its ubiquitous occurrence and important functions, it is unclear how poly P is synthesized or how poly P metabolism is regulated in higher eukaryotes. This work describes a systematic analysis of poly P levels in yeast knockout strains mutated in almost every non-essential gene.

Results: After three consecutive screens, 255 genes (almost 4% of the yeast genome) were found to be involved in the maintenance of normal poly P content. Many of these genes encoded proteins functioning in the cytoplasm, the vacuole or in transport and transcription. Besides reduced poly P content, many strains also exhibited reduced total phosphate content, showed altered ATP and glycogen levels and were disturbed in the secretion of acid phosphatase.

Conclusion: Cellular energy and phosphate homeostasis is suggested to result from the equilibrium between poly P, ATP and free phosphate within the cell. Poly P serves as a buffer for both ATP and free phosphate levels and is, therefore, the least essential and consequently most variable component in this network. However, strains with reduced poly P levels are not only affected in their ATP and phosphate content, but also in other components that depend on ATP or free phosphate content, such as glycogen or secreted phosphatase activity.

Figure 1

All cellular compartments are involved in the maintenance of poly P levels. Categorization of the 255 genes important for poly P levels by using the GO Slim terminology to: (a) cellular component, (b) a biological process and (c) a molecular function. Categories that were significantly overrepresented (P ≤ 0.05; red bars) are marked by an asterisk. Only the 15 most abundant categories are shown (all other genes are summarized as 'other').

Figure 2

Clustering of all 255 genes found in this poly P screen resolved distinct groups with similar poly P profiles. (a) Hierarchical clustering (uncentered Pearson correlation, complete linkage) of all log₂-transformed data (relative to the wild-type). Clusters significantly enriched (P ≤ 0.05) for any GO Slim category are marked by colored bars and branches. (b) Average poly P values (log₂-transformed, relative to the wild type) for all genes in the four distinct clusters in (a) at the four time-points.

Figure 3

Many strains with altered poly P levels are also affected in their total phosphate content, ATP levels, rAPase activity and glycogen accumulation. All measurements (OD₆₀₀, total phosphate content, ATP levels, rAPase activity and glycogen content) in the 30 strains most affected in their poly P levels are given relative to the wild type (log₂-transformed). The data were hierarchically clustered (uncentered Pearson correlation, average linkage).

Figure 4

Poly P links energy and phosphate metabolism in a 'phosphate-energy-network'. Enzymatic reactions between the main phosphate/energy pools, ATP and poly P (shown by black arrows) are (at least partly) hypothesized. Flow to different organic phosphate/energy pools is indicated by the gray arrows. In contrast to ATP, poly P is less directly interconnected with the other phosphate pools and, therefore, its levels fluctuate more.