This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-a-acetylation (NTA) and e-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

ABSTRACT

Glutathione synthetase activity (EC 6.3.2.3) was analysed in ammonium sulfate precipitates of extracts l'rom photohetevotrophically grown cells of Nicotiana tabactm L. cv. Samsun by determination of glutathione as its monobromobimane derivative. Maximal enzyme activity was obtained at pH 8.0–9.0 in Tris‐HCl and CHES as buffer systems. The enzyme showed an absolute requirement for Mg2+ and was slightly stimulated by K+. When Mg2+ was replaced by Mn2+ less synthetase activity was observed, and above 30 mM Mn2+ no activity was found. The enzyme was specific for glycine (KM = 0.308 mM). No product formation was observed with ß‐alanine and γy‐aminobutyrate using substrate conccntrations of 10 mM. The apparent KM values for γ‐glutamylcysteine and γ‐glutamyl‐l‐α‐aminobutyrate were, respectively, 0.022 and 0.033 mM. By chloroplast Isolation ca 24% of the total glutathione synthetase activity of the cells could be shown to be localized in the chloroplasts, the rest being attributed to the cytoplasm of the tobacco cells.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism ofArabidopsisseeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.