Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Effect of Several Nutrients and Environmental Conditions on Intracellular Melatonin Synthesis in Saccharomyces cerevisiae.

Melatonin is a bioactive compound that is present in fermented beverages and has been described to be synthesized by yeast during alcoholic fermentation. The aim of this study was to assess the capacity of intracellular and extracellular melatonin production by different Saccharomyces strains from diverse food origin and to study the effects of different fermentation parameters, such as sugar and nitrogen concentration, temperature or initial population, on melatonin production using a synthetic grape must medium. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry. Intracellular melatonin synthesis profile did not present differences between yeast strains. However, extracellular melatonin production depended on the yeast origin. Thus, we suggest that melatonin production and secretion during the different yeast growth phases follows a species-specific pattern. Other parameters that affected the fermentation process such as sugar content and low temperature had an impact on intracellular melatonin production profile, as well as the melatonin content within the cell. This study reports the effect of several conditions on the melatonin synthesis profile, highlighting its possible role as a signal molecule.

Melatonin and glycolytic protein interactions are related to yeast fermentative capacity.

Melatonin is an indole amine that interacts with some proteins in mammals, such as calreticulin, calmodulin or sirtuins. In yeast, melatonin is synthetized and interacts with glycolytic proteins during alcoholic fermentation in Saccharomyces cerevisiae. Due to its importance as an antioxidant molecule in both Saccharomyces and non-Saccharomyces yeasts, the aim of this study was to determine the intracellular and extracellular synthesis profiles of melatonin in four non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora uvarum, Starmeralla bacillaris and Metschnikowia pulcherrima) and to confirm whether glycolytic enzymes can also interact with this molecule in non-conventional yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced in a similar pattern in all non-Saccharomyces yeast, with M. pulcherrima and S. bacillaris being the highest producers. However, melatonin only bound to proteins in two non-conventional yeasts, S. bacillaris and T. delbrueckii, which specifically had higher fermentative capacities. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes, but an RNA-binding protein, the elongation alpha factor, which is related to mitochondria, was also identified. This study reports for the first time the interaction of melatonin with proteins inside non-Saccharomyces yeast cells. These results reinforce the possible role of melatonin as a signal molecule, likely related to fermentation metabolism and provide a new perspective for understanding its role in yeast.

Glycolytic Proteins Interact With Intracellular Melatonin in Saccharomyces cerevisiae.

Melatonin is a bioactive compound that is present in fermented beverages and synthesized by yeast during alcoholic fermentation. Many studies have shown that melatonin interacts with some mammalian proteins, such as sirtuins or orphan receptor family proteins. The aim of this study was to determine the intracellular synthesis profile of melatonin in Saccharomyces cerevisiae and to identify the proteins that may interact with this molecule in yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced intracellularly in the lag phase of yeast growth and was exported to the extracellular media during the stationary phase. During this period, melatonin was bound to six proteins with molecular weights from 55 to 35 kDa. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes. An RNA-binding protein was also identified, the elongation alpha factor, which is related to mitochondria. This study reports for the first time the interaction of melatonin and proteins inside yeast cells. These results highlight the possible role of melatonin as a signal molecule and provide a new perspective for understanding its role in yeast.

Determination of melatonin by a whole cell bioassay in fermented beverages.

Melatonin is a bioactive compound that is present in fermented beverages, such as wine and beer, at concentrations ranging from picograms to nanograms per mL of product. The purpose of this study was to optimize a novel fluorescent bioassay for detecting melatonin based on a cell line that contains the human melatonin receptor 1B gene and to compare these results with LC-MS/MS as a reference method. Conditions that could affect cell growth and detection (cell number per well, stimulation time, presence or absence of fetal bovine serum and adhesion of cells) were tested in the TANGO® cell line. Food matrices (wine and grape must) could not be directly used for the cell line due to low response. Therefore, for the determination of melatonin in food samples, an extraction procedure was required before conducting the assay. We demonstrated an improvement in melatonin determination by the cell-based bioassay due to increased sensitivity and specificity and improved quantification in complex matrices. Therefore, this method is a good alternative to determine melatonin content in some food samples, especially for those containing very low melatonin levels.