Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p.

Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the two S. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase. Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.

Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity.

Schizosaccharomyces pombe rho1(+) is required for maintenance of cell integrity and polarization of the actin cytoskeleton. However, no other effector besides the (1,3)beta-D-glucan synthase enzyme has been identified in S. pombe. We have further investigated if rho1(+ )signalling could be also mediated by the two protein kinase C homologues, pck1p and pck2p. We show in this study that both kinases interact with rho1p and rho2p only when bound to GTP, as most GTPase effectors do. Interestingly, the interaction was mapped in a different part of the proteins than in Saccharomyces cerevisiae Pkc1p. Thus, active rho1p binds to the amino-terminal region of the pcks where two HR1 motifs are located, and binding to the GTPase dramatically stabilizes the kinases. Detailed biochemical analysis suggests that pck2p is more important in the regulation of the enzyme (1-3)beta-D-glucan synthase. Thus, overexpression of pck2(+), but not pck1(+), caused a general increase in cell wall biosynthesis, mainly in beta-glucan, and (1-3)beta-D-glucan synthase activity was considerably augmented. When this activity was separated into soluble and membrane fractions and reconstituted, the increase caused by pck2(+) overexpression was exclusively detected in the membrane component. We also show that both protein kinase C homologues are required for the maintenance of cell integrity. pck1delta and pck2delta strains present a number of defects related to the cell wall, indicating that this structure might be co-ordinately regulated by both kinases. In addition, pck2p, but not pck1p, seems to be involved in keeping cell polarity. Genetic evidence indicates that both pck1(+) and pck2(+) interact with cps1(+) and gls2(+), two genes similar to S. cerevisiae FKS1 and FKS2 that encode membrane subunits of the (1-3)beta-D-glucan synthase. pck1(+ )also showed a genetic interaction with ras1(+) and ral1(+) suggesting the existence of a functional link between both signalling pathways.