CGL160-mediated recruitment of the coupling factor CF1 is required for efficient thylakoid ATP synthase assembly,photosynthesis, and chloroplast development in Arabidopsis

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CF_O) and a soluble coupling factor (CF₁). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CF_O and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF₁-CF_O assembly. In plants that lacked AtCGL160N, CF₁-CF_O content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF₁ subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF₁ subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF₁-β subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF₁ subcomplex, which is critical for the modulation of CF₁-CF_O activity and photosynthesis.

The green-lineage specific N-terminal domain of CGL160 recruits coupling factor 1 and its lack affects chloroplast development, photosynthesis and thylakoid ATP synthase assembly in Arabidopsis.

IN A NUTSHELL Background: Thylakoid ATP synthases are impressive molecular engines that harness the light-driven proton gradient to generate ATP during photosynthesis. Their molecular mode of operation and atomic structure have been elucidated, but their assembly process is still under investigation. Specific auxiliary factors assist in ATP synthase assembly and prevent the accumulation of dead-end products or deleterious intermediates. CGL160 is one such factor and consists of a membrane and an N-terminal domain. The membrane domain of CGL160 is distantly related to bacterial Atp1 proteins, which are also present in cyanobacteria. Previous studies demonstrated that CGL160 promotes efficient formation of the membranous c-ring of thylakoid ATP synthases in Arabidopsis thaliana. Question: What is the function of the green lineage-specific N-terminal domain of CGL160 in thylakoid ATP synthase assembly, and what is the evolutionary relationship between CGL160 and Atp1? Findings: Here, we showed that the N-terminal domain of CGL160 is required for the late steps in thylakoid ATP synthase assembly and recruits the stromal ATP synthase intermediate coupling factor CF₁. The assembly step is critical for chloroplast development in the dark, ATP synthase activity, and photosynthesis in A. thaliana. We also revealed that Atp1 from the cyanobacterium Synechocystis spec PCC 6803 could functionally replace the membrane domain of CGL160 in A. thaliana. These results indicated that Atp1 operates in c-ring assembly in cyanobacteria and that CGL160 evolved from its cyanobacterial ancestor Atp1. However, CGL160 acquired an additional function in linking a soluble ATP synthase intermediate to a membranous subcomplex. Next steps: The next steps are to identify all auxiliary factors required for the assembly of thylakoid ATP synthases and to understand their precise function in ATP synthase formation. Detailed knowledge of the factors and the assembly process could provide elegant strategies for adjusting proton circuits and altering the ATP budget in crops or other photosynthetic organisms.     

The central effects of morphine on fetal breathing movements in the fetal sheep

The fetal respiratory and electrocortical effects of 0.6 microgram to 600 micrograms of morphine, administered into the lateral cerebral ventricle, have been studied in chronically catheterised, unanaesthetized fetal sheep at 115-135 days gestation. Morphine at 0.6 microgram had no effect on breathing movements or electrocorticographic activity, and at 6 micrograms induced a period of apnoea (43-122 min) but had no effect on electrocortical activity. Intravenous naloxone (2 mg bolus and infusion of 2 mg/kg/h for 2 h) to the fetus had no effect on this apnoea. Morphine at 60 micrograms induced an initial period of apnoea (30-65 min) followed by episodic but significantly deep breathing movements with no effect on electrocortical activity and at 600 micrograms induced an initial period of apnoea (22-95 min) which was followed by deep, irregular and continuous (126-302 min) breathing movements. During the apnoea electrocortical activity initially remained cyclic, but as apnoea progressed there was a gradual reduction in the voltage of the electrocorticogram to a low voltage state. Intravenous naloxone (2 mg bolus and infusion of 2 mg/kg/h for 2 h) reversed both the respiratory and electrocortical effects. The hyperventilation was also inhibited by hypoxia. Naloxone alone had no effect on fetal breathing activity.     

Differential ultracentrifugation enables deep plasma proteomics through enrichment of extracellular vesicles

Human plasma is a rich source of biomedical information and biomarkers. However, the enormous dynamic range of plasma proteins limits its accessibility to mass spectrometric (MS) analysis. Here, we show that enrichment of extracellular vesicles (EVs) by ultracentrifugation increases plasma proteome depth by an order of magnitude. With this approach, more than two thousand proteins are routinely and reproducibly quantified by label-free quantification and data independent acquisition (DIA) in single-shot liquid chromatography tandem mass spectrometry runs of less than one hour. We present an optimized plasma proteomics workflow that enables high-throughput with very short chromatographic gradients analyzing hundred samples per day with deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples. Finally, we test the workflow on clinical biobank samples from malignant melanoma patients in immunotherapy to demonstrate the improved proteome coverage supporting the potential for future biomarker discovery.     

Structure and mechanism of action of an inverting mutant sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Br?nsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure 3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the active site. First, the anomeric oxygen atom of the hydrolysis product (beta-N-acetylneuraminic acid) and four water molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310 moves to make a strong hydrogen bond to one of the bound water molecules.