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We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI [Purnelle B., Skala, J., Van Dijck, L. & Goffeau, A. (1992) Yeast 8, 977-986] encodes UDP-glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP. Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies. In particular, the amino acid sequence motifs involved in the binding of glucose 1-phosphate and UDP-Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi-copy expression of YKL248 resulted in a 40-fold increase in UGPase activity. This gene was, therefore, renamed UGP1. Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability. Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted. This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild-type gene under the control of the glucose-repressible galactose-inducible promoter. The effects of overproducing UGPase on the cell metabolism and morphology were carbon-source dependent. On glucose medium, the 40-fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP-Glc, with no significant effect on growth. In contrast, on galactose, the 40-fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3-5-fold increase in the concentrations of UDP-Glc, UDP-Gal and galactose 1-phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation. Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium. Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5-10% wild-type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium. However, the 10-fold reduction of UGPase activity induced a multi-budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell-wall beta-glucan content. All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP-Glc in yeast metabolism.

KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genesHelene M a r t i n, Ad i I ia Dag kessa ma ns ka ia, Ga I i na Sa t c ha ns ka, Nathalie Dallies and Jean Franqois The KNR4 gene, originally isolated by complementation of a K9 killer-toxinresistant mutant displaying reduced levels of both 1,3-/?-glucan and 1,3-pglucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants. In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of P-glucan and mannan. In all cases, this effect was accompanied b y a strong reduction in mRNA levels corresponding to CHS7, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS7 and RHO7, two genes encoding the catalytic subunit and a regulatory component of 1,3-P-glucan synthase, respectively. Overexpression of KNR4 also inhibited expression of CHS genes in wild-type strains and in two other cwh mutants, whose sensitivity to calcofluor white was not suppressed by this gene. The physiological relevance of the KNR4 transcriptional effect was addressed in two different ways. In a wild-type strain exposed t o a-factor, overexpression of this gene inhibited CHSl induction and delayed shmoo formation, two events which are triggered in response to the pheromone, whereas it did not affect bud formation and cell growth in a chs1 chs2 double mutant. A chimeric protein made by fusing green fluorescent protein t o the C terminus of Knr4p which fully complemented a knr4A mutation was found to localize in patches a t presumptive bud sites in unbudded cells and a t the incipient bud site during bud emergence. Taken together, these results demonstrate that KNR4 has a regulatory role in chitin deposition and in cell wall assembly. A mechanism by which this gene affects expression of CHS genes is proposed.Keywords : Saccharomyces cerevisiae, cell wall, chitin, KNR4, localization INTRODUCTIONYeasts and fungi are surrounded by a thick cell wall which accounts for 25 O/O of the dry mass (Valentin et al., 1987; Fleet, 1991; Dallies et al., 1998). The cell wall is essential for maintenance of cell shape and offers protection against harmful environmental conditions. It is a dynamic structure which can adapt to different physiological states (conjugation, sporulation, station- ary phase, etc.) and morphological changes (i.e. pseudohyphal and agar-invasive growth; Girneno et al., 1992;Roberts & Fink, 1994). It is composed of P-glucans and mannoproteins, which account for about 80-95 % of wall dry mass, and of smaller amounts of chitin (2%) and lipids (3-5%) (for a review, see .