Lev Alexandrovitich Okorokov

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

CaThi is a thionin-like peptide isolated from fruits of Capsicum annuum, which has strong antimicrobial activity against bacteria, yeasts and filamentous fungi, and induced reactive oxygen species (ROS) in fungi. ROS are molecules that appear in the early stages of programmed cell death or apoptosis in fungi. Due to this fact, in this work we analyzed some events that may be related to process of apoptosis on yeast induced by CaThi. To investigate this possibility, we evaluated phosphatidylserine (PS) externalization, presence of active caspases and the ability of CaThi to bind to DNA in Candida tropicalis cells. Additionally, we investigated mitochondrial membrane potential, cell surface pH, and extracellular H⁺ fluxes in C. tropicalis cells after treatment with CaThi. Our results showed that CaThi induced PS externalization in the outer leaflet of the cell membrane, activation of caspases, and it had the ability for DNA binding and to dissipate mitochondrial membrane potential. In addition, the cell surface pH increased significantly when the C. tropicalis cells were exposed to CaThi which corroborates with ~96% inhibition on extracellular H⁺ efflux. Taking together, these data suggest that this peptide is capable of promoting an imbalance in pH homeostasis during yeast cell death playing a modulatory role in the H⁺ transport systems. In conclusion, our results strongly indicated that CaThi triggers apoptosis in C. tropicalis cells, involving a pH signaling mechanism.

Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H⁺-ATPase. Morphological transition was followed microscopically, and the H⁺-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H⁺-ATPase activity and H⁺ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

V H⁺-ATPase has an important role in a variety of key physiological processes. This enzyme is reversibly activated/partly inactivated by the addition/exhaustion of extracellular glucose. The current model of its regulation assumes the reversible disassembly/reassembly of ∼60–70% of the V1 and V0 membrane complexes, which are responsible for ATP hydrolysis and H⁺ conductance, respectively. The number of assembled complexes determines the pump activity because disassembled complexes are inactive. The model predicts the identical catalytic properties for the activated and semi-active enzymes molecules. To verify the model predictions we have isolated total membranes from yeast spheroplasts that were pre-incubated either with or without glucose. Nitrate treatment of membranes revealed the similar ATPase inhibition for two enzyme states, suggesting that they have identical structures that are essential for ATP hydrolysis. However, H⁺ transport was inhibited more than the ATPase activities, indicating a nitrate uncoupling action, which was significantly higher for the nonactivated enzyme. This finding suggests that the structure of the non-activated enzyme, which is essential for H⁺ transport, is less stable than that of the activated enzyme. Moreover, the glucose activation of the pump increases i) its coupling capacity; ii) its K_M for ATP hydrolysis and ATP affinity for H⁺ transport; iii) the Vmax for H⁺ transport in comparison with the Vmax for ATP hydrolysis and iv) the immune reactivity of catalytic subunit A and regulatory subunit B by 9.3 and 2.4 times, respectively. The protein content of subunits A and B was not changed by extracellular glucose. We propose that instead of the dissociation/reassociation of complexes V1 and V0, changes in the extracellular glucose concentration cause reversible and asymmetrical modulations in the immune reactivity of subunits A and B by their putative biochemical modifications. This response asymmetrically modulates H⁺-transport and ATP hydrolysis, exhibiting distinct properties for the activated versus non-activated enzymes.

This study establishes the role of P_5A-type Cta4 ATPase in Ca²⁺ sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive ⁴⁵Ca²⁺-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca²⁺-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4⁺ gene. Furthermore, a disruption of cta4⁺ resulted in 6-fold increase of intracellular Ca²⁺ levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca²⁺ pumping. These data provide compelling biochemical evidence for a P_5A-type Cta4 ATPase as an essential component of Ca²⁺ transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.

The 450-million-year-old interaction between the majority of land plants and mycorrhizal fungi is one of the most ancient, abundant, and ecologically important symbiosis on earth. The early events in the evolution of mycorrhizal symbioses seem to have involved reciprocal genetic changes in ancestral plants and free-living fungi. new data on the mechanism of action of specific signaling molecules and how it influence and is influenced by the membrane ions fluxes and cytoplasm ion oscillations which integrate the symbiotic ionome are improving our understanding of the molecular bases of the mycorrhization process. This mini-review will highlight topics regarding what is known about the ionome and ionic communication in the arbuscular mycorrhizal symbiosis focusing on the signals involved in the development of symbioses. Here we present an overview integrating the available data with the prospects of the research in the field.