γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.

Edited by Hitoshi Nakatogawa c-Glutamyl kinase (GK; the PRO1 gene product) is a key enzyme in the Saccharomyces cerevisiae proline biosynthesis pathway. Dpro1 cells are more sensitive to various stresses than wild-type cells, suggesting that GK has an alternative function independent of proline biosynthesis. We show that PRO1 genetically interacts with UBP3, which encodes ubiquitin-specific protease, and is required for selective autophagy of ribosomes (ribophagy). Interestingly, yeast cells with PRO1 deletion or expressing inactive GK display a defect for ribophagy but not for nonselective autophagy, indicating that GK activity is indispensable for ribophagy. Gene disruption analysis suggests that ribophagy is important for cell survival during nitrogen starvation.Keywords: autophagy; deubiquitination; ribophagy; ribosome; yeast; c-glutamyl kinase The PRO1 gene encodes c-glutamyl kinase (GK), which is the rate-limiting enzyme that catalyzes the first step reaction in the proline biosynthesis of the yeast Saccharomyces cerevisiae. GK phosphorylates the c-carboxylic group of glutamate and is subjected to feedback inhibition by proline. Expression in cells of proline-insensitive variants of GK, such as D154N-GK and I150T-GK, leads to proline accumulation and tolerance to various stresses, such as freezing, ethanol and heat shock, compared to wild-type cells [1][2][3]. By contrast, PRO1-deleted cells were more sensitive to such stresses than wild-type cells [4,5]. Moreover, genome-wide analysis using a yeast-knockout collection showed that the PRO1 deletion results in high sensitivity to various stresses, but not deletion of the PRO2 gene, which encodes the second step enzyme c-glutamyl phosphate reductase in proline biosynthesis [4,6,7]. These results strongly suggest that GK has a novel function independent of proline biosynthesis.Autophagy is a degradation pathway that is conserved in a wide range of eukaryotic cells. Yeast cells induce autophagy during nutrient starvation. Over 40 autophagy-related (Atg) proteins have been identified in S. cerevisiae. In the autophagy process, cytoplasmic components or organs are engulfed in a double-membrane structure (the autophagosome) and the innermembrane and contents are transported into the vacuoles and degraded there. There are two types of autophagy: nonselective autophagy and selective autophagy. Selective autophagy targets mitochondria, the endoplasmic reticulum (ER) and the nucleus in processes referred to as mitophagy, ER-phagy and nucleophagy, respectively [8,9]. The ribosomes are also a target of selective autophagy of ribosomes (i.e. ribophagy) [10,11].Ribosomes are part of the machinery of protein synthesis and consist of two particles: the small (40S) and large (60S) subunits. The half-life of a mature ribosome is estimated to be several days, indicating that mature ribosomes are highly stable. However, the mature ribosome is degraded by ribophagy during nitrogen starvation. Deubiquitination activity that is dependent on the Ubp3-Bre5 complex is required for A...

No abstract