KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes

Abstract

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 .

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1‐dependent cell wall integrity pathway

“…Unless otherwise stated, the yeast cells were grown at 30uC 828 H. Martin-Yken et al in either YEPD (1% Yeast Extract, 2% Bacto Peptone, 2% glucose) or synthetic minimal (SD) medium (0.17% Yeast Nitrogen Base without amino acids and ammonium, 0.5% ammonium sulphate) and 2% glucose, supplemented with auxotrophic requirements. Calcofluor white, killer toxins K2 and K9 and sorbitol-containing plates were made as described previously (Ram et al, 1994;Martin et al, 1999). Geneticin (G418), caffeine and Congo red were prepared as 10-fold concentrated stock solutions made in sterile water.…”

Structure–function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1–SLT2 pathway in Saccharomyces cerevisiae

“…The cytocidal effect of this toxin is due to pore formation at the tip of growing buds and the release of cellular components through them (Takasuka et al, 1995). We isolated the same gene as a multicopy suppressor of several Calcofluor white hypersensitive mutants (Martin et al, 1999). The knr4∆ mutant has a highly permeable cell wall, shows hypersensitivity to cell wall drugs (caffeine, Calcofluor white, cercosporamide,), and arrests growth at temperatures above 37…”

Recommendations for assigning symbols and names to Neurospora crassa genes now that its genome has been sequenced.

“…The apparently unnecessary C-terminal region of the protein may be involved in additional cellular functions not examined in this study. Support for this possibility can be found in reports suggesting that the role of the S. cerevisiae SM1/KNR4 homologue of COT2 is, most likely, regulatory (Martin et al 1999), and that on the basis of additional analyses in yeasts (Dagkessamanskaia et al 2001), it is conceivable that COT2 participates, via interactions with several proteins, in additional cellular processes. Other alleles of cot-2/ gs-1 have been identified in an additional screen (Seiler and Plamann 2003).…”