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Microbiology 152 (2006), 2635-2649; DOI  10.1099/mic.0.29105-0
© 2006 Society for General Microbiology

KlRHO1 and KlPKC1 are essential for cell integrity signalling in Kluyveromyces lactis

Rosaura Rodicio1,2, Sabrina Koch1, Hans-Peter Schmitz1 and Jürgen J. Heinisch1

1 Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, 49076 Osnabrück, Germany
2 Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33006 Oviedo, Spain

Correspondence
Jürgen J. Heinisch
heinisch{at}biologie.uni-osnabrueck.de

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 1161 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.


Abbreviations: 5-FOA, 5-fluoroorotic acid; GFP, green fluorescent protein

The GenBank/EMBL/DDBJ accession numbers for the KlPKC1 and KlRHO1 sequences reported in this paper are AY129673 and AY129674, respectively.




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M. Kohli, S. Buck, and H.-P. Schmitz
The function of two closely related Rho proteins is determined by an atypical switch I region
J. Cell Sci., April 1, 2008; 121(7): 1065 - 1075.
[Abstract] [Full Text] [PDF]




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