no significant differences in growth between mutant yeast cells on SD agar medium with heat shock (50°C, 3 to 5 h), ethanol (16 to20%), and sorbitol (2.0 to 2.5 M) stresses, although the proline-accumulating cells are more resistant to these stresses than cells expressing wild-type GK
no significant differences in growth between mutant yeast cells on SD agar medium with heat shock (50°C, 3 to 5 h), ethanol (16 to20%), and sorbitol (2.0 to 2.5 M) stresses, although the proline-accumulating cells are more resistant to these stresses than cells expressing wild-type GK
the enzyme has an alternative function independent of proline biosynthesis. Gene PRO1 genetically interacts with UBP3, which encodes ubiquitin-specific protease, and is required for selective autophagy of ribosomes (ribophagy). The enzyme activity is indispensable for ribophagy, ribophagy is important for cell survival during nitrogen starvation
the DELTApro1 cells are more sensitive to various stresses than wild-type cells. Yeast cells with gene PRO1 deletion or expressing inactive enzyme display a defect for ribophagy but not for nonselective autophagy. Yeast strains deficient ribophagy are sensitive to nitrogen starvation
Yeast cells expressing mutant enzymes D154N and I150T accumulate proline and show a higher tolerance to various stresses, including freezing, ethanol, osmotic pressure, and heat shock
residues Asp154 and Ile150 in the Saccharomyces cerevisiae gamma-glutamate kinase are important for allosteric regulation and the affinity with L-proline and L-glutamate, but not with ATP. The PUA domain itself is required for the full display of enzyme activity, but is not involved in the allosteric regulation and the affinity with substrates and proline. The proline-auxotrophic yeast strain BY4741DELTApro1DELTAcar2 that expresses the PUA domain deletion-mutant grows even on proline-free medium, although a growth defect is observed
very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
the mutation results in extreme desensitization to feedback inhibition by L-proline, leading to L-proline overproduction. The relative activity of the variant is 96% in the presence of 100 mM L-proline and 87% in the presence of 400 mM L-proline. The specific activity of the variant is slightly reduced compared with the wild type enzyme
the mutation results in a prominant increase in cell viability after freezing at -20°C compared to the viability of the cells harboring the wild-type PRO1 gene. The altered gamma-glutamyl kinase results in stabilization of the complex with gamma-glutamyl phosphate reductase or has an indirect effect on gamma-glutamyl phosphate reductase activity which leads to an increase in L-proline production in Saccharomyces cerevisiae
site-directed mutagenesis, the mutant enzyme is less sensitive to proline feedback inhibition compared to the wild-type enzyme and shows an increased thermostability
very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
site-directed mutagenesis, the mutant enzyme is less sensitive to proline feedback inhibition compared to the wild-type enzyme and shows an increased thermostability
the mutant is greatly insensitive to feedback inhibition, leading to L-proline overproduction. The relative activity of the variant is 59% in the presence of 100 mM L-proline. The specific activity of the variant is slightly reduced compared with the wild type enzyme
construction of a gene PRO1 disruption mutant, phenotype, detailed overview. Overexpression of wild-type enzyme partially suppresses the phenotype of the DELTApro1 strain, which is deficient in ribophagy
truncated yeast gamma-glutamyl kinase proteins are engineered from which the C-terminal region is deleted. A complementation test in Escherichia coli and yeast and enzymatic analysis of recombinant proteins reveal that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue archaeosine transglycosylase (PUA) domain is essential for gamma-glutamyl kinase activity. 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full enzymatic activity
the D154N mutation results in a prominant increase in cell viability after freezing at -20°C compared to the viability of the cells harboring the wild-type PRO1 gene, method for breeding novel freeze-tolerant yeast strains