Alpha-1-B glycoprotein (A1BG) inhibits sterol-binding and export by CRISP2.

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in various processes, including sperm maturation and cancer progression. They are mostly secreted glycoproteins and share a unique conserved CAP domain. The precise mode of action of these proteins, however, has remained elusive. Saccharomyces cerevisiae expresses three members of this protein family, which bind sterols in vitro and promote sterol secretion from cells. This sterol-binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with alpha-1-B glycoprotein (A1BG), a human plasma glycoprotein that is upregulated in different types of cancers. Here we examined whether the interaction between CRISP proteins and A1BG affects the sterol-binding function of CAP family members. Co-expression of A1BG with CAP proteins abolished their sterol export function in yeast and their interaction inhibits sterol-binding in vitro. We map the interaction between A1BG and CRISP2 to the third of five repeated immunoglobulin-like (Ig) domains within A1BG. Interestingly, the interaction between A1BG and CRISP2 requires magnesium, suggesting that coordination of Mg2+ by the highly conserved tetrad residues within the CAP domain is essential for a stable interaction between the two proteins. The observation that A1BG modulates the sterol-binding function of CRISP2, has potential implications for the role of A1BG and related Ig domain containing proteins in cancer progression and the toxicity of reptile venoms containing CRISP proteins.

Binding of perilipin 3 to membranes containing diacylglycerol is mediated by conserved residues within its PAT domain.

Perilipins (PLINs) constitute an evolutionarily conserved family of proteins that specifically associate with the surface of lipid droplets (LDs). These proteins function in LD biogenesis and lipolysis and help to stabilize the surface of LDs. PLINs are typically composed of three different protein domains. They share an N-terminal PAT domain of unknown structure and function, a central region containing 11-mer repeats that form amphipathic helices, and a C-terminal domain that adopts a 4-helix bundle structure. How exactly these three distinct domains contribute to PLIN function remains to be determined. Here, we show that the N-terminal PAT domain of PLIN3 binds diacylglycerol (DAG), the precursor to triacylglycerol, a major storage lipid of LDs. PLIN3 and its PAT domain alone bind liposomes with micromolar affinity and PLIN3 binds artificial LDs containing low concentrations of DAG with nanomolar affinity. The PAT domain of PLIN3 is predicted to adopt an amphipathic triangular shaped structure. In silico ligand docking indicates that DAG binds to one of the highly curved regions within this domain. A conserved aspartic acid residue in the PAT domain, E86, is predicted to interact with DAG, and we found that its substitution abrogates high affinity binding of DAG as well as DAG-stimulated association with liposome and artificial LDs. These results indicate that the PAT domain of PLINs harbor specific lipid-binding properties that are important for targeting these proteins to the surface of LDs and to ER membrane domains enriched in DAG to promote LD formation.

The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH.

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photosynthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H+ ions are transported in exchange for Ca2+ and Mn2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+, Mn2+ or H+ gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H+ ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H+ transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts.

The human Golgi protein TMEM165 transports calcium and manganese in yeast and bacterial cells.

Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi TMEM165 (transmembrane protein 165), belonging to UPF0016 (uncharacterized protein family 0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca2+- and Mn2+-transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast (Saccharomyces cerevisiae) and in the bacterium Lactococcus lactis Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively autoregulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca2+ and Mn2+ transporters abrogates Ca2+- and Mn2+-induced growth defects, excessive Mn2+ accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca2+ and Mn2+ influxes. We also used the yeast and bacterial systems to evaluate the impact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca2+ and Mn2+, which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations.

The yeast protein Gdt1p transports Mn2+ ions and thereby regulates manganese homeostasis in the Golgi.

The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. More precisely, genetic variants of the human UPF0016 ortholog transmembrane protein 165 (TMEM165) have been linked to congenital disorders of glycosylation (CDG). The Saccharomyces cerevisiae ortholog Gdt1p has been shown to be involved in calcium homeostasis and protein glycosylation. Moreover, plant and bacterial UPF0016 members appear to have putative roles in Mn2+ homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Lactococcus lactis Using Mn2+-induced quenching of Fura-2-emitted fluorescence, we observed that Gdt1p mediates Mn2+ influx, in addition to its previously reported regulation of Ca2+ influx. The estimated Km values of Gdt1p of 15.6 ± 2.6 µm for Ca2+ and 83.2 ± 9.8 µm for Mn2+ indicated that Gdt1p has a higher affinity for Ca2+ than for Mn2+ In yeast cells, we found that Gdt1p is involved in the resistance to high Mn2+ concentration and controls total Mn2+ stores. Lastly, we demonstrated that GDT1 deletion affects the activity of the yeast Mn2+-dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn2+ concentrations. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn2+ homeostasis and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast cells may regulate Golgi intraluminal concentrations of manganese, a key cofactor of many enzymes involved in protein glycosylation.

Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis.

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO2 fixation. Changes in free Ca2+ in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca2+ fluxes in the chloroplasts have been unknown. By employing a Ca2+ reporter-based approach, we identified two chloroplast-localized Ca2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca2+ signal in the chloroplast stroma. BICAT2 mediated Ca2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca2+ ]stroma signal. Conversely, this Ca2+ transient was increased in knockout mutants of BICAT1, which transports Ca2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca2+ transporters.

Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development.

BACKGROUND: The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae. RESULTS: In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p. CONCLUSIONS: The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The knowledge on the important enzyme involved in higher alcohols biosynthesis by S. kudriavzevii could be of scientific as well as of applied interest.

Perilipin 3 promotes the formation of membrane domains enriched in diacylglycerol and lipid droplet biogenesis proteins.

Lipid droplets (LDs) serve as intracellular stores of energy-rich neutral lipids. LDs form at discrete sites in the endoplasmic reticulum (ER) and they remain closely associated with the ER during lipogenic growth and lipolytic consumption. Their hydrophobic neutral lipid core is covered by a monolayer of phospholipids, which harbors a specific set of proteins. This LD surface is coated and stabilized by perilipins, a family of soluble proteins that specifically target LDs from the cytosol. We have previously used chimeric fusion proteins between perilipins and integral ER membrane proteins to test whether proteins that are anchored to the ER bilayer could be dragged onto the LD monolayer. Expression of these chimeric proteins induces repositioning of the ER membrane around LDs. Here, we test the properties of membrane-anchored perilipins in cells that lack LDs. Unexpectedly, membrane-anchored perilipins induce expansion and vesiculation of the perinuclear membrane resulting in the formation of crescent-shaped membrane domains that harbor LD-like properties. These domains are stained by LD-specific lipophilic dyes, harbor LD marker proteins, and they transform into nascent LDs upon induction of neutral lipid synthesis. These ER domains are enriched in diacylglycerol (DAG) and in ER proteins that are important for early steps of LD biogenesis, including seipin and Pex30. Formation of the domains in vivo depends on DAG levels, and we show that perilipin 3 (PLIN3) binds to liposomes containing DAG in vitro. Taken together, these observations indicate that perilipin not only serve to stabilize the surface of mature LDs but that they are likely to exert a more active role in early steps of LD biogenesis at ER subdomains enriched in DAG, seipin, and neutral lipid biosynthetic enzymes.

Potassium supply and homeostasis in the osmotolerant non-conventional yeasts Zygosaccharomyces rouxii differ from Saccharomyces cerevisiae.

Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomyces cerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1∆ trk2∆ cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of growth at low K(+) concentrations and it changed some cell physiological parameters in a way that differs from S. cerevisiae. In contrast to S. cerevisiae, Z. rouxii cells without the TRK1 gene contained less potassium than the control cells and their plasma membrane was significantly hyperpolarized compared with those of the parental strain when grown in the presence of 100 mM KCl. On the other hand, subsequent potassium starvation led to a substantial depolarization which is again different from S. cerevisiae. Plasma-membrane hyperpolarization did not prevent the efflux of potassium from Z. rouxii trk1Δ cells during potassium starvation, and the activity of ZrPma1 is less affected by the absence of ZrTRK1 than in S. cerevisiae. The use of a newly constructed Z. rouxii-specific plasmid for the expression of pHluorin showed that the intracellular pH of the Z. rouxii wild type and the trk1∆ mutant is not significantly different. Together with the fact that Z. rouxii cells contain a significantly lower amount of intracellular potassium than identically grown S. cerevisiae cells, our results suggest that this highly osmotolerant yeast species maintain its intracellular pH and potassium homeostasis in way(s) partially distinct from S. cerevisiae.

Dimerization of an immunoactivating peptide derived from mycobacterial hsp65 using N-hydroxysuccinimide based bifunctional reagents is critical for its antitumor properties.

We have shown previously that a short pentapeptide derived from the mycobacterial heat shock protein hsp65 can be highly activating for the immune system based on its strong reactivity with the early activation antigen of lymphocytes CD69. Here, we investigated an optimal form of presentation of this antigen to the cells of the immune system. Four different forms of the dimerized heptapeptide LELTEGY, and of the control inactive dimerized heptapeptide LELLEGY that both contained an extra UV active glycine-tyrosine sequence, were prepared using dihydroxysuccinimidyl oxalate (DSO), dihydroxysuccinimidyl tartarate (DST), dihydroxysuccinimidyl glutarate (DSG), and dihydroxysuccinimidyl suberate (DSS), respectively. Heptapeptides dimerized through DST and DSG linkers had optimal activity in CD69 precipitation assay. Moreover, dimerization of active heptapeptide resulted in a remarkable increase in its proliferation activity and production of cytokines in vitro. Furthermore, while DST and DSG dimerized heptapeptides both significantly enhanced the cytotoxicity of natural killer cells in vitro, only the DSG dimerized compound was active in suppressing growth of melanoma tumors in mice and in enhancing the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Thus, while the dimerization of the immunoactive peptide caused a dramatic increase in its immunoactivating properties, its in vivo anticancer properties were influenced by the chemical nature of linker used for its dimerization.

From the Uncharacterized Protein Family 0016 to the GDT1 family: Molecular insights into a newly-characterized family of cation secondary transporters.

The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn2+, with an extra reported function in Ca2+ and/or H+ transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca2+ signaling in yeast, and Mn2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles.

Yeast as a Tool for Deeper Understanding of Human Manganese-Related Diseases.

The biological importance of manganese lies in its function as a key cofactor for numerous metalloenzymes and as non-enzymatic antioxidant. Due to these two essential roles, it appears evident that disturbed manganese homeostasis may trigger the development of pathologies in humans. In this context, yeast has been extensively used over the last decades to gain insight into how cells regulate intra-organellar manganese concentrations and how human pathologies may be related to disturbed cellular manganese homeostasis. This review first summarizes how manganese homeostasis is controlled in yeast cells and how this knowledge can be extrapolated to human cells. Several manganese-related pathologies whose molecular mechanisms have been studied in yeast are then presented in the light of the function of this cation as a non-enzymatic antioxidant or as a key cofactor of metalloenzymes. In this line, we first describe the Transmembrane protein 165-Congenital Disorder of Glycosylation (TMEM165-CDG) and Friedreich ataxia pathologies. Then, due to the established connection between manganese cations and neurodegeneration, the Kufor-Rakeb syndrome and prion-related diseases are finally presented.

New Trends in the Uses of Yeasts in Oenology.

The most important factor in winemaking is the quality of the final product and the new trends in oenology are dictated by wine consumers and producers. Traditionally the red wine is the most consumed and more popular; however, in the last times, the wine companies try to attract other groups of populations, especially young people and women that prefer sweet, whites or rosé wines, very fruity and with low alcohol content. Besides the new trends in consumer preferences, there are also increased concerns on the effects of alcohol consumption on health and the effects of global climate change on grape ripening and wine composition producing wines with high alcohol content. Although S. cerevisiae is the most frequent species in wines, and the subject of most studies, S. uvarum and hybrids between Saccharomyces species such as S. cerevisiae×S. kudriavzevii and S. cerevisiae×S. uvarum are also involved in wine fermentations and can be preponderant in certain wine regions. New yeast starters of non-cerevisiae strains (S. uvarum) or hybrids (S. cerevisiae×S. uvarum and S. cerevisiae×S. kudriavzevii) can contribute to solve some problems of the wineries. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts, while fulfilling the requirements of the commercial yeasts, such as a good fermentative performance and aromatic profiles that are of great interest for the wine industry. In this review, we will analyze different applications of nonconventional yeasts to solve the current winemaking demands.

Differences in Enzymatic Properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum Alcohol Acetyltransferases and Their Impact on Aroma-Active Compounds Production.

Higher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavor and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalyzed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g., S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate esters formation when compared to S. cerevisiae. Here, we aimed to analyze the enzymes encoded by the ATF1 and ATF2 genes from S. kudriavzevii (SkATF1, SkATF2) and S. uvarum (SuATF1, SuATF2). The heterologous expression of the individual ATF1 and ATF2 genes in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. Particularly, an increase of 2-phenylethyl acetate production by the strains that harbored ATF1 and ATF2 genes from S. kudriavzevii and S. uvarum was observed. When grown with individual amino acids as the nitrogen source, the strain that harbored SkATF1 showed particularly high 2-phenylethyl acetate production and the strains with introduced SkATF2 or SuATF2 revealed increased production of isobutyl acetate, isoamyl acetate, and 2-phenylethyl acetate compared to the reference strains with endogenous ATF genes. The alcohol acetyltransferase activities of the individual Atf1 and Atf2 enzymes measured in the cell extracts of the S. cerevisiae atf1 atf2 iah1 triple-null strain were detected for all the measured substrates. This indicated that S. kudriavzevii and S. uvarum Atf enzymes had broad range substrate specificity as S. cerevisiae Atf enzymes. Individual Atf1 enzymes exhibited markedly different kinetic properties since SkAtf1p showed c. twofold higher and SuAtf1p c. threefold higher K m for isoamyl alcohol than ScAtf1p. Together these results indicated that the differences found among the three Saccharomyces species during the aroma-active acetate ester formation may be due, to some extent, to the distinct properties of Atf enzymes.

Saccharomyces kudriavzevii and Saccharomyces uvarum differ from Saccharomyces cerevisiae during the production of aroma-active higher alcohols and acetate esters using their amino acidic precursors.

Higher alcohols and acetate esters are important flavour and aroma components in the food industry. In alcoholic beverages these compounds are produced by yeast during fermentation. Although Saccharomyces cerevisiae is one of the most extensively used species, other species of the Saccharomyces genus have become common in fermentation processes. This study analyses and compares the production of higher alcohols and acetate esters from their amino acidic precursors in three Saccharomyces species: Saccharomyces kudriavzevii, Saccharomyces uvarum and S. cerevisiae. The global volatile compound analysis revealed that S. kudriavzevii produced large amounts of higher alcohols, whereas S. uvarum excelled in the production of acetate esters. Particularly from phenylalanine, S. uvarum produced the largest amounts of 2-phenylethyl acetate, while S. kudriavzevii obtained the greatest 2-phenylethanol formation from this precursor. The present data indicate differences in the amino acid metabolism and subsequent production of flavour-active higher alcohols and acetate esters among the closely related Saccharomyces species. This knowledge will prove useful for developing new enhanced processes in fragrance, flavour, and food industries.