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.
Laboratorium voor Moleculaire Celbiologie, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Flanders, Belgium.
M
M
Vanhalewyn
F
F
Dumortier
G
G
Debast
S
S
Colombo
P
P
Ma
J
J
Winderickx
P
P
Van Dijck
J M
JM
Thevelein
D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D002874:Chromosome Mapping; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015398:Signal Transduction / Q000235:genetics
D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D005947:Glucose