KlRHO1 and KlPKC1 are essential for cell integrity signalling in Kluyveromyces lactis.
Cell integrity in yeasts is ensured by a rigid cell wall whose synthesis is triggered by a MAP kinase-mediated signal-transduction cascade. Upstream regulatory components of this pathway in Saccharomyces cerevisiae involve a single protein kinase C, which is regulated by interaction with the small GTPase Rho1. Here, two genes were isolated which encode these proteins from Kluyveromyces lactis (KlPKC1 and KlRHO1). Sequencing showed ORFs which encode proteins of 1,161 and 208 amino acids, respectively. The deduced proteins shared 59 and 85 % overall amino acid identities, respectively, with their homologues from S. cerevisiae. Null mutants in both genes were non-viable, as shown by tetrad analyses of the heterozygous diploid strains. Overexpression of the KlRHO1 gene under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and K. lactis. On the other hand, a similar construct with KlPKC1 did not show a pronounced phenotype. Two-hybrid analyses showed interaction between Rho1 and Pkc1 for the K. lactis proteins and their S. cerevisiae homologues. A green fluorescent protein (GFP) fusion to the C-terminal end of KlPkc1 located the protein to patches in the growing bud, and at certain stages of the division process also to the bud neck. N-terminal GFP fusions to KlRho1 localized mainly to the cell surface (presumably the cytoplasmic side of the plasma membrane) and to the vacuole, with some indications of traffic from the former to the latter. Thus, KlPkc1 and KlRho1 have been shown to serve vital functions in K. lactis, to interact in cell integrity signalling and to traffic between the plasma membrane and the vacuole.
Rodicio
Rosaura
R
Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33006 Oviedo, Spain.|Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, 49076 Osnabrück, Germany.
Koch
Sabrina
S
Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, 49076 Osnabrück, Germany.
Schmitz
Hans-Peter
HP
Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, 49076 Osnabrück, Germany.
Heinisch
Jürgen J
JJ
Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, 49076 Osnabrück, Germany.
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020043:Genes, Essential; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014617:Vacuoles / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins