Global screening of genes essential for growth in high-pressure and cold environments: searching for basic adaptive strategies using a yeast deletion library

Abstract

Microorganisms display an optimal temperature and hydrostatic pressure for growth. To establish the molecular basis of piezo- and psychroadaptation, we elucidated global genetic defects that give rise to susceptibility to high pressure and low temperature in Saccharomyces cerevisiae. Here we present 80 genes including 71 genes responsible for high-pressure growth and 56 responsible for low-temperature growth with a significant overlap of 47 genes. Numerous previously known cold-sensitive mutants exhibit marked high-pressure sensitivity. We identified critically important cellular functions: (i) amino acid biosynthesis, (ii) microautophagy and sorting of amino acid permease established by the exit from rapamycin-induced growth arrest/Gap1 sorting in the endosome (EGO/GSE) complex, (iii) mitochondrial functions, (iv) membrane trafficking, (v) actin organization mediated by Drs2-Cdc50, and (vi) transcription regulated by the Ccr4-Not complex. The loss of EGO/GSE complex resulted in a marked defect in amino acid uptake following high-pressure and low-temperature incubation, suggesting its role in surface delivery of amino acid permeases. Microautophagy and mitochondrial functions converge on glutamine homeostasis in the target of rapamycin (TOR) signaling pathway. The localization of actin requires numerous associated proteins to be properly delivered by membrane trafficking. In this study, we offer a novel route to gaining insights into cellular functions and the genetic network from growth properties of deletion mutants under high pressure and low temperature.

Figure 1.—

Growth properties of the wild-type strain BY4742 and the trp1Δ mutant. Cells were cultured under the pressures shown at 24° (A) or at the temperatures shown at 0.1 MPa (B). The OD_600ap value was measured immediately after decompression. The OD_600ap values were converted into OD₆₀₀ values using the formula described in materials and methods.

Figure 2.—

Representation of growth defects of deletion mutants on a pressure–temperature diagram. The OD₆₀₀ values are normalized to those of the wild-type strain BY4742 (A) in Table 1. Eighty mutants were classified into 11 categories: (B) amino acid biosynthesis, (C) microautophagy, (D) mitochondrial functions, (E) actin organization and bud formation, (F) membrane trafficking, (G) transcription and mRNA degradation, (H) ribosome function, (I) inositol phosphate (Ins-P) metabolism, (J) chromatin maintenance, (K) stress response, and (L) unknown function. All results are merged in M. Data are represented as mean values with standard deviations obtained from three independent experiments.

Figure 3.—

The EGO complex is essential for growth at high pressure and low temperature. Cells containing the specified plasmids were cultured at 0.1 MPa and 24°, 25 MPa and 24°, or 0.1 MPa and 15°. The OD₆₀₀ values were measured after 20 hr of culture. pEGO1, low-copy plasmid containing EGO1 driven by its own promoter; pEGO3, low-copy plasmid containing EGO3 driven by its own promoter; pL137, low-copy plasmid containing GTR1 driven by its own promoter; pCDV987, containing GTR2 driven by its own promoter. Data are represented as mean values with standard deviations obtained from three independent experiments.

Figure 4.—

Cell growth of the EGO complex mutants following release from high-pressure and low-temperature incubation. (A) Cells were cultured in SC medium at 0.1 MPa and 24° following release from a 10-hr incubation at 0.1 MPa and 24°, 25 MPa and 24°, or 0.1 MPa and 15°. (B) Cells were cultured in glutamine- and glutamate-enriched SC medium at 0.1 MPa and 24° following release from a 10-hr incubation at 0.1 MPa and 24°, 25 MPa and 24° or 0.1 MPa and 15°. (C) Cells were cultured in 5× SC medium at 0.1 MPa and 24° following release from a 10-hr incubation at 0.1 MPa and 24°, 25 MPa and 24°, or 0.1 MPa and 15°. Data are confirmed by duplicate experiments.

Figure 5.—

Amino acid uptake following release from high-pressure and low-temperature incubation. Cells of the wild type, ego1Δ, gtr1Δ, and cdc50Δ were cultured at 0.1 MPa and 24°, 25 MPa and 24°, or 0.1 MPa and 15° for 5 hr followed by the measurement of uptake of histidine, lysine, and leucine at 0.1 MPa and 24° using ³H-labeled substrates (see materials and methods). Data are represented as mean values with standard deviations obtained from three to seven independent experiments.

Figure 6.—

Cell growth of the EGO/GSE complex mutants with or without nutriment auxotrophy. The wild-type and the EGO/GSE complex mutants with (A) auxotrophy for leucine, lysine, histidine, and uracil or (B) prototrophy were cultured at 0.1 MPa and 24°, 25 MPa and 24°, or 0.1 MPa and 15°. The prototrophic strains were cultured in SC medium in the absence of leucine, lysine, histidine, and uracil for maintenance of the plasmids. The OD₆₀₀ values were measured after 20 hr of culture. (C) The OD₆₀₀ values are normalized to those of the wild-type strain BY4742 as shown in Figure 2. Data are represented as mean values with standard deviations obtained from three independent experiments.

Figure 7.—

Physical and genetic interactions among the genes identified in this study. The interactions are registered in the Saccharomyces Genome Database. Note that MSS4 is an essential gene for yeast viability and was not analyzed in this study. However, it is indicated for its importance. Arrows, genetic interactions, i.e., synthetic lethality and synthetic slow growth; solid bars, physical interactions. Ins-P, inositol phosphate.