Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

ABSTRACTMany metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Intracellular Acidification Suppresses Synaptic Vesicle Mobilization in the Motor Nerve Terminals

Intracellular protons play a special role in the regulation of presynaptic processes, since the functioning of synaptic vesicles and endosomes depends on their acidification by the H+-pump. Furthermore, transient acidification of the intraterminal space occurs during synaptic activity. Using microelectrode recording of postsynaptic responses (an indicator of neurotransmitter release) and exo-endocytic marker FM1-43, we studied the effects of intracellular acidification with propionate on the presynaptic events underlying neurotransmitter release. Cytoplasmic acidification led to a marked decrease in neurotransmitter release during the first minute of a 20-Hz stimulation in the neuromuscular junctions of mouse diaphragm and frog cutaneous pectoris muscle. This was accompanied by a reduction in the FM1-43 loss during synaptic vesicle exocytosis in response to the stimulation. Estimation of the endocytic uptake of FM1-43 showed no disruption in synaptic vesicle endocytosis. Acidification completely prevented the action of the cell-membrane permeable compound 24-hydroxycholesterol, which can enhance synaptic vesicle mobilization. Thus, the obtained results suggest that an increase in [H+]in negatively regulates neurotransmission due to the suppression of synaptic vesicle delivery to the sites of exocytosis at high activity. This mechanism can be a part of the negative feedback loop in regulating neurotransmitter release.

Intramelanocytic Acidification Plays a Role in the Antimelanogenic and Antioxidative Properties of Vitamin C and Its Derivatives

Although vitamin C (VC, L-ascorbic acid) has been widely used as a skin lightening agent for a long time, the mechanism by which it inhibits melanogenesis remains poorly understood. It is well-documented that the intramelanocytic pH is an important factor in regulating tyrosinase function and melanosome maturation. The activity of tyrosinase, the rate-limiting enzyme required for melanin synthesis, is generally minimal in an acidic environment. Given that VC is an acidic compound, we might speculate that the intracellular acidification of melanocytes induced by VC likely reduces melanin content through the suppression of tyrosinase activity. The results of this study reveal that treatment of melanocytes with VC or its derivatives, magnesium ascorbyl phosphate (MAP) and 3-O-ethyl-L-ascorbic acid (AAE), resulted in significant decreases in the tyrosinase activity and melanin content and in the levels of intracellular reactive oxygen species (ROS), indicating that VC and its derivatives possess antimelanogenic and antioxidative activities. Western blotting analysis indicated that VC, MAP, and AAE exert their antimelanogenic activity by inhibiting the tyrosinase activity rather than by downregulating the expression of melanogenic proteins such as tyrosinase, premelanosome protein 17 (Pmel17) and microphthalmia-associated transcription factor (MITF). Further, we found that the reduced tyrosinase activity of melanocytes treated with VC or its derivatives could be reactivated following intracellular neutralization induced by ammonium chloride (NH4Cl) or concanamycin A (Con A). Finally, we examined the expression of sodium-dependent VC transporter-2 (SVCT-2) using western blotting and qPCR, which revealed that there was a significant increase in the expression of SVCT-2 in melanocytes following treatment with VC. VC-mediated intracellular acidification was neutralized by phloretin (a putative SVCT-2 inhibitor) in a dose-dependent manner. Taken together, these data show that VC and its derivatives suppress tyrosinase activity through cytoplasmic acidification that potentially results from enhanced VC transmembrane transport via the VC transporter SVCT-2.

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis

ABSTRACTPyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved.Mycobacterium tuberculosislacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification whenM. tuberculosisis exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility toM. tuberculosisunder neutral and even alkaline growth conditions. Furthermore, we find that wild-typeM. tuberculosisdisplays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain ofM. tuberculosisthat expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

Microbial Growth under Supercritical CO2

ABSTRACTGrowth of microorganisms in environments containing CO2above its critical point is unexpected due to a combination of deleterious effects, including cytoplasmic acidification and membrane destabilization. Thus, supercritical CO2(scCO2) is generally regarded as a sterilizing agent. We report isolation of bacteria from three sites targeted for geologic carbon dioxide sequestration (GCS) that are capable of growth in pressurized bioreactors containing scCO2. Analysis of 16S rRNA genes from scCO2enrichment cultures revealed microbial assemblages of varied complexity, including representatives of the genusBacillus. Propagation of enrichment cultures under scCO2headspace led to isolation of six strains corresponding toBacillus cereus,Bacillus subterraneus,Bacillus amyloliquefaciens,Bacillus safensis, andBacillus megaterium. Isolates are spore-forming, facultative anaerobes and capable of germination and growth under an scCO2headspace. In addition to these isolates, severalBacillustype strains grew under scCO2, suggesting that this may be a shared feature of spore-formingBacillusspp. Our results provide direct evidence of microbial activity at the interface between scCO2and an aqueous phase. Since microbial activity can influence the key mechanisms for permanent storage of sequestered CO2(i.e., structural, residual, solubility, and mineral trapping), our work suggests that during GCS microorganisms may grow and catalyze biological reactions that influence the fate and transport of CO2in the deep subsurface.