Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast.
Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.
Bartish
Galyna
G
School of Life Sciences, Södertörns högskola, Huddinge, Sweden.
Moradi
Hossein
H
Nygård
Odd
O
D049109:Cell Proliferation; D002470:Cell Survival; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009995:Osmosis; D020652:Peptide Elongation Factor 2 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020652:Peptide Elongation Factor 2; D013912:Threonine