Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd^- phenotype), while other substitutions block kinase activation (Gcn^- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn^- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd^- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.     

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.     

Dark starvation and chloroplast function

Summary Mature spinach plants were held in the dark for several days. The photochemical activities and the activity of some enzymes related to NADP reduction were follwed in the chloroplasts isolated from leaves after dark starvation. Photosystem-II, measured by reduction of DPIP, remained stable during 6 days of darkening. The decrease of NADP reduction which appeared after 2 days of starvation was found to be due to protein autolysis rather than inactivation of the photosystems. The stability of photosystem-I was demonstrated by reactivation of NADP reduction after addition of purified ferredoxin and ferredoxin-NADP-reductase. After 4 days of starvation the restoration of the NADP reduction required in addition another, low-molecular-weight factor. From the isolation procedure and from its properties this factor is assumed to be identical with FRS. However, even in the presence of FRS only half of the total activity is restored after 7 days. The activity of the NADP-reducing system is restored in vivo when plants kept for 7 days in the dark are again illuminated.Abbreviations NADP nicotinamide-adenine-dinucleotide phosphate - DPIP 2,6-dichlorophenolindophenol - DCMU (3,4-dichlorophenyl)-1,1-dimethylurea - FRS ferredoxin-reducing-substance