RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP.
We have previously shown that a conserved glycine at position 82 of the yeast RAS2 protein is involved in the conversion of RAS proteins from the GDP- to the GTP-bound form. We have now investigated the role of glycine 82 and neighbouring amino acids of the distal switch II region in the physiological mechanism of activation of RAS. We have introduced single and double amino acid substitutions at positions 80-83 of the RAS2 gene, and we have investigated the interaction of the corresponding proteins with a yeast GDP dissociation stimulator (SDC25 C-domain). Using purified RAS proteins, we have found that the SDC25-stimulated conversion of RAS from the GDP-bound inactive state to the GTP-bound active state was severely impaired by amino acid substitutions at positions 80-81. However, the rate and the extent of conversion from the GDP- to the GTP-bound form in the absence of dissociation factor was unaffected. The insensitivity of the mutated proteins to the dissociation factor in vitro was paralleled by an inhibitory effect on growth in vivo. The mutations did not significantly affect the interaction of RAS with adenylyl cyclase. These findings point to residues 80-82 as important determinants of the response of RAS to GDP dissociation factors. This suggests a molecular model for the enhancement of nucleotide release from RAS by such factors.
Verrotti
A C
AC
Differentiation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Créchet
J B
JB
Di Blasi
F
F
Seidita
G
G
Mirisola
M G
MG
Kavounis
C
C
Nastopoulos
V
V
Burderi
E
E
De Vendittis
E
E
Parmeggiani
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005800:Genes, Fungal; D005838:Genotype; D005998:Glycine; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D020668:rap GTP-Binding Proteins; D018631:ras Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium; D005998:Glycine