Identification of regulatory residues of the yeast adenylyl cyclase.
We have attempted to identify amino acid residues of the yeast adenylyl cyclase that are involved in the regulation of its activity, by isolating adenylyl cyclase-linked spontaneous mutations capable of suppressing the temperature-sensitive phenotype of ras1- ras2-ts1 strains. We previously identified a mutated adenylyl cyclase in which a single point mutation, called CR14, led to the replacement of threonine 1651 with isoleucine. We have now investigated the biological effects of CR14, and of other mutations that cause the replacement of threonine 1651 by distinct amino acids. We have observed that the response of adenylyl cyclase to Ras can be either enhanced or attenuated, without significant effects on the steady-state level of the former enzyme in vivo, depending on the amino acid side chain at position 1651. Therefore, this residue identifies a regulatory region on the adenylyl cyclase molecule. We have also taken advantage of the attenuation of adenylyl cyclase function caused by the replacement of threonine 1651 with aspartic acid to isolate intragenic suppressor mutations. We have identified several point mutations, leading to single amino acid substitutions, individually capable of reactivating the attenuated adenylyl cyclase. The corresponding amino acid changes are located within a relatively small region, including residues 1331, 1345, 1348 and 1374. This region could be physiologically involved in the negative control of the carboxy-terminal catalytic domain.
Feger
G
G
Differentiation Structures Programme, European Molecular Biology Laboratory, Heidelberg, FRG.
De Vendittis
E
E
Vitelli
A
A
Masturzo
P
P
Zahn
R
R
Verrotti
A C
AC
Kavounis
C
C
Pal
G P
GP
Fasano
O
O
D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D015825:Chromosomes, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors; D005838:Genotype; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes; D000262:Adenylyl Cyclases