Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae

Abstract

Hydrostatic pressure in the range of 15 to 25 MPa was found to cause arrest of the cell cycle in G(1) phase in an exponentially growing culture of Saccharomyces cerevisiae, whereas a pressure of 50 MPa did not. We found that a plasmid carrying the TAT2 gene, which encodes a high-affinity tryptophan permease, enabled the cells to grow under conditions of pressure in the range of 15 to 25 MPa. Additionally, cells expressing the Tat2 protein at high levels became endowed with the ability to grow under low-temperature conditions at 10 or 15 degrees C as well as at high pressure. Hydrostatic pressure significantly inhibited tryptophan uptake into the cells, and the Tat2 protein level was down-regulated by high pressure. The activation volume associated with tryptophan uptake was found to be a large positive value, 46.2 +/- 3.85 ml/mol, indicating that there was a net volume increase in a rate-limiting step in tryptophan import. The results showing cell cycle arrest in G(1) phase and down-regulation of the Tat2 protein seem to be similar to those observed upon treatment of cells with the immunosuppressive drug rapamycin. Although rapamycin treatment elicited the rapid dephosphorylation of Npr1 and induction of Gap1 expression, hydrostatic pressure did not affect the phosphorylation state of Npr1 and it decreased the level of Gap1 protein, suggesting that the pressure-sensing pathway may be independent of Npr1 function. Here we describe high-pressure sensing in yeast in comparison with the TOR-signaling pathway and discuss an important factor involved in adaptation of organisms to high-pressure environments.

FIG. 1

Hydrostatic pressure reduces the growth rate in wild-type strain YPH499. Exponentially growing cells were subjected to a hydrostatic pressure of 0.1, 15, 25, or 50 MPa in hydrostatic chambers. The chambers were opened at 5-h intervals in the period up to 15 h after the start of incubation, and then the cells were spread on YPD agar plates and incubated for a few days at 0.1 MPa. Data presented are mean values with SD of results from three independent experiments.

FIG. 2

Hydrostatic pressure causes cell cycle arrest in G₁ phase in the wild-type strain YPH499. Exponentially growing cells were exposed to a hydrostatic pressure of 0.1, 15, 25, or 50 MPa for 5 or 10 h as indicated at the upper right in each graph. After decompression, samples were prepared for flow cytometry. Propidium iodide-labeled cells (approximately 10⁵ cells) were analyzed using FACSCalibur.

FIG. 3

TAT2 and TRP1 confer high-pressure growth. Using cultures of exponentially growing cells as inocula (the initial cell density [N₀] was approximately 10⁶ cells/ml), several transformants were grown at 0.1 MPa (A), 15 MPa (B), 25 MPa (C), or 50 MPa (D) for 24 h in SCv medium. All data are presented as ratios of the cell density at 24 h (N₂₄) to the cell density at N₀ and are mean values with SD of results from three independent experiments.

FIG. 4

Expression of TAT2 confers high-pressure growth as well as low-temperature growth. Strains FAA4 (YEplac195) and FAB19 (YEplac195::TAT2) were grown under various pressure conditions at 24°C (A to C) or under various temperature conditions at 0.1 MPa (A, D, E, and F) in SCv medium.

FIG. 5

Hydrostatic pressure inhibits and down-regulates tryptophan uptake. Tryptophan uptake was assayed as described in Materials and Methods. Hydrostatic pressure was applied at the 10-min point in each experiment. Strain FAA4 was incubated at 25 MPa for 0 h (A), 2 h (B), or 5 h (C), and subsequently the tryptophan uptake assay was performed. Strain FAB33 was incubated at 25 MPa for 0 h (D), 2 h (D), or 5 h (F), and subsequently the tryptophan uptake assay was performed. Data presented are mean values with SD of results from three independent experiments.

FIG. 6

Hydrostatic pressure down-regulates intracellular levels of Tat2 protein. Exponentially growing cells of several transformants were exposed to a pressure of 25 MPa for 2, 5, or 10 h or treated with rapamycin (200 ng/ml), and Western blot analysis was performed. (A) Down-regulation of the Tat2 protein in strain FAB33 (YEplac195::2HA-TAT2); (B) down-regulation of the Tat2 protein in strain FAB59 (YCplac33::2HA-TAT2); (C) phosphorylation state of the Npr1 protein (Npr1^p) in strain FAE12 (pRS103); (D) down-regulation of the Gap1 protein in strain FAF6 (pPL257). Exp., experiment; Rap, rapamycin.