KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis.
Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade. In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p. This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively. We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family. The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S. cerevisiae. Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence. Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K. lactis. Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway. Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and in K. lactis. The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway.
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 26.12, D-40225 Düsseldorf, Germany.
Anja
A
Lorberg
Hans-Peter
HP
Schmitz
Ute
U
Gengenbacher
Jürgen J
JJ
Heinisch
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics*; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; C097993:Pkc1 protein, Trichoderma reesei; D011493:Protein Kinase C