Microbiology (1 996), 142, 1765-1 773 Printed in Great Britain 1 Departrn nt of Molecula Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Laboratoriurn Vlaardingen, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands 2 U ni lever Research The Cdc25 protein of Saccharomyces cerevisiae is required for normal glucose transport Herman H. W. Sillj6,’ Eelko G. ter Schure,l Arie J. Verkleij,’ Johannes Boonstral and C. The0 Verrips1#2 Author for correspondence: Herman H. W. SilljC. Tel: +31 30 2532598. Fax: +31 30 2513655. e-mail : herman@emsal .biol.ruu.nl The essential CDC25 gene product of Saccharomyces cerewisiae is the most upstream known component of the RASIadenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its CAMP metabolism, it is concluded that this effect on glucose transport is independent of CAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased CAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the g I ucose transporters. Keywords : Saccharom_yces cerevisiae, cdc25 mutants, glucose metabolism, glucose uptake, continuous culture I INTRODUCTION The RAS/adenylate cyclase signal transduction pathway plays an important role in the regulation of metabolism and in cell cycle control in the yeast Saccharomyes cerevisiae (Broach, 1991 ; Thevelein, 1992, 1994). Addition of fermentable carbon sources to a yeast culture growing on a non-fermentable carbon source results in the activation of the RAS/adenylate cyclase pathway, which leads to a transient rise in the intracellular CAMP level (Thevelein, 1992). This in turn results in the liberation of the CAMP- dependent protein kinase A (PKA) (Toda et al., 1987a,b) from the regulatory subunit (Toda et al., 1987a) resulting in the activation of PKA. PKA in turn phosphorylates key enzymes of carbon metabolism, for example fructose- 1,6-bisphosphatase (Rittenhouse et al., 1987) and phos- phofructokinase 2 (Fransois etal., 1984), resulting in a net increase in glycolytic activity versus gluconeogenic ac- tivity. The most upstream known activator of the RAS/ adenylate cyclase pathway is the Cdc25 protein (Broek et al., 1987; Camonis et al., 1986; Van Aelst et al., 1991). Cdc25 is a guanine-nucleotide-exchange factor (GEF) replacing GDP bound to RAS protein by GTP, resulting in the activation of RAS. GTP-activated RAS in turn activates adenylate cyclase, which catalyses the conversion of ATP into CAMP, stimulating PKA activity. The nucleotide sequence of the CDC25 gene codes for a large protein of 1588 or 1589 amino acid residues (Broek et al., 1987 ; Camonis et al., 1986). Gene disruption and deletion experiments have identified three functional domains of the Cdc25 protein. The amino-terminal half (the a- domain), containing an SH3 domain (Rodaway et al., .............................................................. I ........ ..... I.............. ............................................................... Abbreviations: cdc, cell division cycle; PKA, protein kinase A; G6P, glucose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; GEF, guanine- 1989), is required for efficient sporulation, gluconeogenic functions and glucose-induced CAMP signalling, the nucleotide-exchangefactor. carboxy-terminal half (the pl -domain), containing the 0002-0721 0 1996 SGM 1765