A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.     

Regulation of the subcellular localization of cyclic AMP-dependent protein kinase in response to physiological stresses and sexual differentiation in the fission yeast Schizosaccharomyces pombe

We describe regulation of the subcellular localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) regulatory (Cgs1p) and catalytic (Pka1p) subunits in the fission yeast Schizosaccharomyces pombe in response to physiological stresses and during sexual differentiation as determined by fluorescence microscopy of the Cgs1-green fluorescent protein (GFP) and Pka1-GFP fusion proteins, respectively. In wild-type S. pombe cells cultured to log phase under normal growth conditions, Cgs1p and Pka1p are concentrated in the nucleus and more diffusely present in the cytoplasm. Nuclear localization of both proteins is dependent on cAMP, since in cells lacking adenylate cyclase they are detectable only in the cytoplasm. In cells lacking Cgs1p or both Cgs1p and adenylate cyclase, Pka1p is concentrated in the nucleus, demonstrating a role for Cgs1p in the nuclear exclusion of Pka1p. Nuclear-cytoplasmic redistribution of Cgs1p and Pka1p is triggered by growth in glucose-limited or hyperosmotic media and in response to stationary-phase growth. In addition, both proteins are excluded from the nucleus in mating cells undergoing karyogamy and subsequently concentrated in postmeiotic spores. Cgs1p is required for subcellular redistribution of Pka1p induced by growth in glucose-limited and hyperosmotic media and during karyogamy but is not required for Pka1p redistribution triggered by stationary-phase growth or for the enrichment of Pka1p in spores. Our results demonstrate that PKA localization is regulated by cAMP and regulatory subunit-dependent and -independent mechanisms in S. pombe.     

Hsp90 protein in fission yeast Swo1p and UCS protein Rng3p facilitate myosin II assembly and function

The F-actin-based molecular motor myosin II is involved in a variety of cellular processes such as muscle contraction, cell motility, and cytokinesis. In recent years, a family of myosin II-specific cochaperones of the UCS family has been identified from work with yeasts, fungi, worms, and humans. Biochemical analyses have shown that a complex of Hsp90 and the Caenorhabditis elegans UCS domain protein UNC-45 prevent myosin head aggregation, thereby allowing it to assume a proper structure. Here we demonstrate that a temperature-sensitive mutant of the fission yeast Hsp90 (Swo1p), swo1-w1, is defective in actomyosin ring assembly at the restrictive temperature. Two alleles of swo1, swo1-w1 and swo1-26, showed synthetic lethality with a specific mutant allele of the fission yeast type II myosin head, myo2-E1, but not with two other mutant alleles of myo2 or with mutations affecting 14 other genes important for cytokinesis. swo1-w1 also showed a strong genetic interaction with rng3-65, a gene encoding a mutation in the fission yeast UCS domain protein Rng3p, which has previously been shown to be important for myosin II assembly. A similar deleterious effect was found when myo2-E1, swo1-w1, and rng3-65 were pharmacologically treated with geldanamycin to partially inhibit Hsp90 function. Interestingly, Swo1p-green fluorescent protein is detected at the improperly assembled actomyosin rings in myo2-E1 but not in a wild-type strain. Yeast two-hybrid and coimmunoprecipitation analyses verified interactions between Rng3p and the myosin head domain as well as interactions between Rng3p and Swo1p. Our analyses of Myo2p, Swo1p, and the UCS domain protein Rng3p establish that Swo1p and Rng3p collaborate in vivo to modulate myosin II function.