γ‐Glutamyl kinase is involved in selective autophagy of ribosomes in Saccharomyces cerevisiae

Abstract

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...

Selective Autophagy of the Protein Homeostasis Machinery: Ribophagy, Proteaphagy and ER-Phagy

“…Using an image-based screen of yeast mutants, the Ubp3 and Bre5 de-ubiquitinase complex was shown to be specifically required for degradation of the large ribosomal subunit (Kraft et al, 2008). In follow-up studies, the Ubp3 and Bre5 binding partners, Cdc48 and Ufd3, were identified as additional players in this process, as well as γ-Glutamyl kinase (Ossareh-Nazari et al, 2010;Tatehashi et al, 2016). The Ubp3 complex de-ubiquitinates lysine 74 on Rpl25, the same residue that is ubiquitinated by the ribosome associated E3 ligase Ltn1.…”

Proteasome Activator Blm10 Regulates Transcription Especially During Aging

“…1E ). Tatehashi et al showed previously that ATG7-deficient yeast is defective in autophagy [ 34 ] . These results demonstrate that the core histones can be degraded by the Blm10-proteasome in the non-replicating yeast, suggesting that Blm10 promotes the transcription-coupled degradation of the core histones.…”

Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases

“…Considering the lack of specific receptors for selective autophagy targeting ribosomes and molecular structure of ribosomes, the relevant research on ribophagy has been progressing slowly and majority of studies focus on yeast. By applying Saccharomyces cerevisiae , a study conducted by Tatehashi et al 85 revealed that γ-glutamine kinase could interact with Ubp3 and participate in ribophagy other than involving in the biosynthesis of proline. Ribophagy was obviously blocked when suppressing the expression of γ-glutamine kinase via knocking out its encoding gene PRO1 , indicating its importance in ribophagy induction 85 .…”

“…By applying Saccharomyces cerevisiae , a study conducted by Tatehashi et al 85 revealed that γ-glutamine kinase could interact with Ubp3 and participate in ribophagy other than involving in the biosynthesis of proline. Ribophagy was obviously blocked when suppressing the expression of γ-glutamine kinase via knocking out its encoding gene PRO1 , indicating its importance in ribophagy induction 85 . Meanwhile, downstream kinases Sch9 and Rim15 of mTOR complex 1 (mTORC1) was demonstrated to play an essential role in the autophagic degradation of ribosomes in budding yeast 86 .…”