0000000000115071
AUTHOR
Ottavio Fasano
Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein
AbstractRas proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserve…
TheSCH9 protein kinase mRNA contains a long 5′ leader with a small open reading frame
The SCH9 yeast gene, that was previously identified as a suppressor of cdc25 and ras1- ras2-ts temperature-sensitive mutants, encodes a putative protein kinase that positively regulates the progression of yeast cells through the G1 phase of the cell cycle. We have determined the structure of the SCH9 transcription unit, using primer extension and S1 mapping techniques. The corresponding mRNA included an unusually long 5' region of more than 600 nucleotides preceding the major open reading frame (ORF). While the latter corresponded to a protein of 824 amino acids, an upstream open reading frame (uORF) within the 5' leader could potentially encode a 54 amino acid peptide. To investigate the r…
Phosphorylation of an Overexpressed Yeast Ras2 Protein During the G1 Phase of the Cell Cycle
RAS proteins regulate growth and differentiation in evolutionarily distant systems such as vertebrates and yeast (for reviews, see Tamanoi, 1988; Gibbs and Marshall, 1989; Broach and Deschenes, 1990). At the moleular level, a key function of the yeast RAS1 and RAS2 proteins (collectively referred to as RAS) is to positively regulate the production of cyclic AMP at the onset of the G1 phase of the cell cycle (Toda et al., 1985; De Vendittis et al., 1986). At this stage, RAS proteins are transiently activated by the noncovalent binding of a GTP molecule. Reversal of the effect occurs by the hydrolytic splitting of the ’γ-phosphate of GTP, that leaves a functionally inactive RASGDP complex, th…
Energetic aspects of intramolecular coupling between the nucleotide binding site and the distal switch II region of the yeast RAS2 protein
AbstractWe have studied the interaction of the yeast RAS2 protein with guanine nucleotides using energetic parameters for the dissociation of RAS·nucleotide complexes. The results indicated that a Gly → Ser substitution at position 82 led to an altered interaction with GppNHp and, to a lesser extent, also with GDP. It was also possible to conclude that structural perturbation of Gly82 can stimulate nucleotide release by decreasing the energetic barrier for nucleotide dissociation. This, together with the observation that residues 80 and 81 are involved in the response of RAS to nucleotide exchange factors without affecting GDP binding per se, suggests a potential mechanism for exchange fact…