Yeast Genetics and Molecular Biology 1996
Madison, Wisconsin
August 1996

Name: Jacobus Albertyn .
Mailing Address: Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, 9300, South Africa. (From 1 July 1996), Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, 77251, USA.
Email Address: KOBUSA@WWG3.UOVS.AC.ZA
Phone and Fax numbers: +27-51-4012875 , +27-51-4482004

The regulation of the FPS1 glycerol fasilitator in Saccharomyces cerevisiae

J. Albertyn1,3, F.C.W. Sutherland1, S.G. Kilian1, B.A. Prior1, S. Hohmann2 . 1) Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa. 2) Department of General and Marine Microbiology, University of Goteborg, Medicinaregaten 9C, S-41319, Goteborg, Sweden. 3) Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, 77251, USA.

Wild type cells of Saccharomyces cerevisiae W303-1A pregrown at either 0.96 aw or 0.998 aw (NaCl or sucrose) showed biphasic kinetics of glycerol uptake. The Km and Vmax values derived by graphical analysis of Eadie-Hofstee plots were the same at both water activities. An osmotic upshock from 0.998 aw to 0.980 aw (NaCl), 0.97 aw (NaCl) or 0.96 aw (NaCl or sucrose) resulted in a sharp decrease in glycerol uptake rate in the wild type strain and after 2 minutes only passive diffusion was evident from Eadie-Hofstee plots, suggesting osmotic regulation independent of both the osmoticum used and the degree of osmotic shock. Conversely, a downshock from 0.96 aw to 0.998 aw resulted in the rapid activation of the system. The glycerol uptake rate in the fps1 deletion mutant was not affected by either an up-or downshock, indicating that osmoregulation of glycerol uptake is dependent on Fps1. The fps1 deletion mutant transformed with FPS1 on a multicopy vector (YEp FPS1) reacted in the same way to an osmotic up-or downshock as the wild type. The strain with the C-terminal truncation (YEpFPS1DC) and the strain with the combined N-and-C- termini truncations (YEpFPS1DNC), previously shown to lack transport function, did not react to an osmotic upshock. The strain with the N-terminal truncation (YEpFPS1DN) retains its ability to transport glycerol but does not close upon an osmotic upshock. These data indicate that the N-terminal plays an important role in osmotic regulation of Fps1. The cells transformed with the glpF gene encoding the glycerol transport system in E. coli did not show a significant decrease in glycerol uptake after cells were subjected to an osmotic upshock. GlpF is therefore not osmotically regulated in Saccharomyces cerevisiae.