KIRHO1 and KIPKC1 are essential for cell integrity signalling in Kluyveromyces lactis

Abstract

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 (KIPKC1 and KIRHO1). 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 KIRHO1 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 KIPKC1 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 KIPkc1 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 KIRho1 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, KIPkc1 and KIRho1 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.