The Escherichia coli proB gene corrects the proline auxotrophy of Saccharomyces cerevisiae pro1 mutants.
We constructed plasmids carrying the Escherichia coli proB gene that encodes gamma-glutamyl kinase, under the control of the yeast GAL1 promoter. This construction was carried out with both the wild-type proB+ gene and a mutant allele, proB74, that specifies an enzyme resistant to feedback inhibition by proline. Yeast pro1 mutants harboring these plasmids are proline prototrophs. We conclude that the pro1 mutation results in a deficiency in the gamma-glutamyl kinase activity in Saccharomyces cerevisiae. Expression of the proB74 allele in yeast resulted in enhanced resistance to the proline analogue L-azetidine-2-carboxylate and in a 2.4-fold elevation of the intracellular free proline levels. This result suggests that gamma-glutamyl kinase is the rate limiting step in proline biosynthesis in yeast.
Orser
C S
CS
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
Goodner
B W
BW
Johnston
M
M
Gelvin
S B
SB
Csonka
L N
LN
D000483:Alleles; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005838:Genotype; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics*; D017851:Phosphotransferases (Carboxyl Group Acceptor); D010957:Plasmids; D011392:Proline / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D011392:Proline; D010770:Phosphotransferases; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase; D004262:DNA Restriction Enzymes