TY - JOUR

T1 - The HUP1 gene product of Chlorella kessleri

T2 - H+/glucose symport studied in vitro

AU - Opekarová, Miroslava

AU - Caspari, Thomas

AU - Tanner, Widmar

PY - 1994/8/24

Y1 - 1994/8/24

N2 - An in vitro system was established to measure secondary active transport mediated by plant H+ symporters. For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes. After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative). Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene. The Km value for D-glucose uptake was 5 x 10(-5) M. The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters. In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation. The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.

AB - An in vitro system was established to measure secondary active transport mediated by plant H+ symporters. For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes. After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative). Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene. The Km value for D-glucose uptake was 5 x 10(-5) M. The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters. In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation. The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.

KW - Chlorella

KW - Hydrogen-Ion Concentration

KW - Kinetics

KW - Monosaccharide Transport Proteins

KW - Schizosaccharomyces

KW - Thermodynamics

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/0005-2736(94)90214-3

DO - 10.1016/0005-2736(94)90214-3

M3 - Article

C2 - 8075129

VL - 1194

SP - 149

EP - 154

JO - Biochimica et Biophysica Acta (BBA)

JF - Biochimica et Biophysica Acta (BBA)

SN - 0006-3002

IS - 1

ER -