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Candida glabrata is considered a major opportunistic fungal pathogen of humans and has emerged as a leading cause of nosocomial fungal infections. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that Candida can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of these fungi. Our recent study (Mota et al., 2015) supported this view by demonstrating that acetic acid influences C. glabrata behavior in biofilm formation, antifungal drug resistance and phagocytosis; and suggesting a potential role of putative carboxylate transporters on these processes. In order to extend our studies and provide a comprehensive view of the C. glabrata biofilms’ response to alternative carbon sources and antifungal treatment, we performed comparative transcriptomics analyses using RNA-sequencing. Our data support the view that adaptative responses of Candida cells to the types of carbon source present in host niches affects the virulence of these fungal cells through multifarious mechanisms (Brown et al., 2014). Finally, elucidating the effect of local nutrients and pH environment on drug resistance can potentially provide new and effective treatment strategies for C. glabrata infections such as vaginal candidiasis., info:eu-repo/semantics/publishedVersion

Este estudo tem como objetivo identificar a presença de uma anomalia de mercado conhecida por efeito janeiro nos mercados desenvolvidos e emergentes. Para esta análise foram utilizados os índices MSCI World e MSCI Emergents Markets dos últimos 20 anos (1994-2015). Foi adotada a metodologia de power ratio e a regressão linear com recurso a variáveis dummy. Os resultados do estudo apontam para a presença do efeito janeiro quer nos mercados desenvolvidos quer nos mercados emergentes, apresentando-se como mais persistente no tempo nos mercados desenvolvidos e mais forte nos mercados emergentes. Para os dois tipos de mercado observa-se, igualmente, um declínio do efeito janeiro, no tempo.

The cell plasma membrane contains transmembrane proteins capable of recognizing and transporting a variety of compounds, such metabolites, nutrients and signaling molecules. Given the impermeability of the plasma membrane, the function, regulation and malfunction of these transporters is crucial for the cell. The intracellular traffic and turnover of transporters is highly regulated and well-studied in fungi and animal cells (Mukherjee et al., 2006; Lauwers et al., 2009; Steinbusch et al., 2011; Scheller, 2013; Liaunardy-Jopeace and Gay, 2014). Our group uses Jen1p, a lactate transporter of Saccharomyces. cerevisiae, as a model membrane cargo to study aspects of the mechanisms controlling the endocytic turnover of transporters in response to physiological signals and stress. Glucose triggers rapid endocytosis of Jen1p via its ubiquitylation by Rsp5p ubiquitin ligase (Paiva et al., 2002; Paiva et al., 2009), in a process that requires the arrestin-like adaptor Rod1 (Becuwe et al., 2012). Recently, it was described that when Rod1 is active, it is mostly localized at the trans-Golgi network (Becuwe and Leon, 2014). However, it remains largely unknown which Jen1 intracellular domains are targeted by Rod1p and Rsp5p, or how these trans-acting factors are recruited to Jen1p. Here, we performed domain swap experiments between Jen1p and other well-studied transporters, which respond to distinct endocytic signals, in order to further understand the specificity of interactions of membrane cargoes with factors of the ubiquitylation machinery. We will present the genetic strategy employed, as well as, data regarding the functional characterization of chimeric transporters., info:eu-repo/semantics/publishedVersion

Poster apresentado no "XXXIX Portuguese Genetics Conference", em Braga, Portugal, 2015, The monocarboxylate transporter Jen1 of the yeast Saccharomyces cerevisiae has proven to be an excellent model system for genetically dissecting mechanisms that regulate trafficking of a eukaryotic plasma membrane protein, according to physiological constraints. Glucose triggers a rapid endocytosis of Jen1 being the main signal ubiquitylation through Rsp5p ubiquitin ligase (Paiva et al., 2002; Paiva et al., 2009) and the arrestin-like adaptor Rod1 (Becuwe et al., 2012). In an attempt to identify domains that are important for the subcellular localization, activity and turnover of Jen1, domain swap experiments were carried out. It was used a novel strategy, based on the gap repair technique to construct 9 chimeric proteins by the replacement of either the N- or the C-terminal half of Jen1 by the N- or the C-terminal half (or both terminals) of the Gap1 general aminoacid transporter, Fur4 uracil transporter and Hxt6 hexose transporter. Gap1 and Fur4 are regulated by ammonium and excess of substrate, respectively (Hein et al., 1995) in contrast to Hxt6 that are regulated by glucose just as Jen1. Each gene was also C-terminally fused with the ORF of the green fluorescent protein (GFP). Here, we will present the successful genetic strategy and the characterization of two chimeras at the protein and subcellular levels., This work was supported by FCT/MEC through Portuguese funds (PIDDAC) - SFRH/BD/86221/2012 and PEst-OE/BIA/UI4050/2014, info:eu-repo/semantics/publishedVersion

PAIVA, S. V. Didemnidae (Tunicata: Ascidiacea) dos French Frigate Shoals. Fortaleza, 2015. 114 f. Dissertação (Mestrado em Ciências Marinhas Tropicais) - Instituto de Ciências do Mar, Universidade Federal do Ceará, Fortaleza, 2015. Ascidians are a diverse class of benthic marine invertebrates, and about 20% of the described species belong to the family Didemnidae. This is due especially to the high diversity of the genus Didemnum, with over 200 known species. Although widely distributed, little is known about this fauna from many regions of the world, such as the Hawaiian archipelago. French Frigate Shoals (Kanemilohai) is the first and the largest atoll located North of the Hawaii mountain chain and belongs to Papahãhanaumokuãkea Marine National Monument, which is the largest US conservation area and one of the largest protected areas in the world. In 2006 an expedition was conducted to French Frigate Shoals as part of the Census of Marine Life program, in order to assess the diversity of cryptic organisms. The ascidians were collected at 18 stations through SCUBA diving or snorkelling. The specimens were collected manually on the reef or from the breakdown of dead coral blocks brought on board. Later they were sent to the Laboratório de Ecologia Animal, Instituto de Ciências do Mar - UFC, where are preserved in 70% ethanol solution. A total of 127 specimens of sea squirts from the Didemnidae family were collected. Of these species, only D. cf. candidum, D. granulatum, D. perlucidum, D. listerianum, D. simile and L. rufus were previously registered to Hawaii, so this work is also the first record of 19 more species to the Hawaiian Archipelago. This work is the main contribution to ascidians of Hawaii. As ascídias constituem uma classe diversa de invertebrados marinhos bentônicos, e cerca de 20% das espécies descritas pertencem à família Didemnidae. Isso se deve, especialmente, à alta diversidade do gênero Didemnum, com mais de 200 espécies conhecidas. Embora sejam animais amplamente distribuídos, pouco se sabe a respeito dessa fauna em várias regiões do mundo, como no arquipélago Havaiano. French Frigate Shoals (Kanemilohai) é o primeiro e o maior atol ao norte da cadeia montanhosa do Havaí e pertence ao Papahãhanaumokuãkea Marine National Monument, que é a maior unidade de conservação dos Estados Unidos e uma das maiores áreas de proteção ambiental do mundo. Em 2006 foi realizada uma expedição aos French Frigate Shoals como parte do “Census of Marine Life”, com o objetivo de inventariar a diversidade de organismos crípticos. As ascídias foram coletadas em 18 estações através de mergulho autônomo utilizando SCUBA ou mergulho livre. Os espécimes foram coletados manualmente no recife ou a partir da quebra de blocos de corais mortos trazidos a bordo. Posteriormente foram encaminhadas ao Laboratório de Ecologia Animal, do Instituto de Ciências do Mar - UFC, onde encontram-se preservados em solução de etanol 70%. Durante a campanha foram coletados 127 espécimes de ascídias da família Didemnidae. Assim, esse trabalho tem como objetivo realizar o inventário das espécies de ascídias da família Didemnidae desse atol havaiano. Das espécies encontradas, 11 são espécies ainda não descritas. Dessas espécies, apenas D. cf. candidum, D. granulatum, D. perlucidum, D. listerianum, D. simile e L. rufus haviam sido registradas para o Havaí, portanto, este trabalho também realiza o primeiro registro de mais 19 espécies para o arquipélago. O presente trabalho é a principal contribuição para as ascídias do arquipélago havaiano.

The S. cerevisiae membrane protein Jen1 is a monocarboxylate-proton symporter which after the addition of glucose to lactic acid-grown cells triggers loss of Jen1p activity and repression of JEN1 gene expression (Paiva et al., 2002; Andrade et al., 2001). This downregulation of the permease is dependent on phosphorylation and ubiquitylation mechanisms, both of which are dependent on proteins interacting with the Jen1p. To identify the proteins involved in this process we developed a system combined structure prediction and the yeast two-hybrid system to overcome the difficulties associated to measuring protein interactions with membrane proteins. Measuring protein interactions between membrane proteins or between membrane proteins and other proteins has proved to be very difficult. The commonly used yeast two-hybrid systems are inefficient for membrane proteins since the interaction must take place in the cell nucleus between soluble proteins. An advantage of the strategy used in our approach is that the screen is unbiased, i.e. contains all proteins in the yeast proteome. The structure of Jen1p predicts that it contains three cytosolic domains that would be expected participate in physiological protein interactions, important for regulation. We have cloned the three predicted cytosolic domains (1, 2 and 3) as bait fusions in the and screened a S. cerevisiae genomic library (James et al. 1996) after that we identified tree genes CIN5, PYC2 e SRO7. Based on this new approach, we hope to establish a reference library in yeast that may be used for many different purposes.

The emergence of probiotics and prebiotics has revived the importance of short-chain fatty acids (SCFAs) associated to colonic and systemic health improvement. Although biosynthesis and degradation of SCFAs and other short-chain carboxylic acids, such as lactate, pyruvate or citrate are well understood, the transport of these acids is still a matter of discussion. The presence of SCFAs transporters in cellular membranes is ubiquitous, displaying great level of homology among Bacteria, Archaea and Eukaryotes, indicating the ancient nature of these transporters and their high level of conservation. The mechanism of substrate uptake of these transporters including specificity, kinetics and bioenergetic studies is a field poorly explored. This work constitutes a first approach to establish the mode of action of the SCFA transporters specifically those belonging to the YaaH family. The YaaH family, (TC# 2.A.96, http://www.tcdb.org/tcdb) is presumed to be a family of acetate transporters. Its members possess 6 putative transmembrane span domains and are spread by the 3 domains of life: Bacteria, Eukaryotes and Archaea. This work aims at studying the YaaH protein from E. coli as well as it homologues from the yeast Saccharomyces cerevisiae (ScAdy2) and the fungi Aspergillus nidulans (AcpA). We have constructed a disrupted E. coli strain in the yaaH gene showing that the yaaH mutant cells are compromised for the uptake of the labelled acetic acid in comparison with the isogenic wt strain. This is the first experimental data that demonstrates the physiological role of the yaaH gene in the transport of acetate in bacteria. Using the yeast and fungi strains we were able to measure the kinetic parameters associated with these transporters and assign a specificity profile to this family. These transporters are specific primarily to acetate and are inhibited by other short chain acids such as benzoic, formic, propionic and butyric acids., FCT PhD grant SFRH/BD/61530/2009 (JSP)

The intracellular trafficking of plasma membrane proteins, such as receptors and transporters, in eukaryotic cells is a highly regulatedprocess. Changing environment conditions (nutrients, substrates, hormones) can trigger endocytosis of unwanted transporters. The monocarboxylate transporter Jen1p of the yeast Saccharomyces cerevisiae has proven to be an excellent model system forgenetically dissecting mechanisms that regulate trafficking of a eukaryotic PM protein, according to physiological constraints. Ourgroup has demonstrated that glucose acts as a signal to induce endocytic down-regulation of Jen1 within minutes, a processdependent both on phosphorylation and ubiquitylation (Paiva et al, 2002; Paiva et al, 2009).In an attempt to identify domains that are important for the subcellular localization, activity and turnover of Jen1p, domain swapexperiments were carried out. The hybridtransporter genes also carry a C-terminal fusion with the ORF of the green fluorescent protein (GFP), which enabled the in vivomicroscopic investigation of the trafficking, membrane localization and turnover of Jen1p protein. This strategy is ideal to identify whether the Jen1 terminals include molecular determinants necessary and sufficient for glucoseelicitedendocytosis and sorting to endosomes. Studies regarding the rates of endocytosis and/or direct sorting of the chimerictransporters, under various physiological conditions, will be presented. Uptake assays using radiolabeled lactic acid were also employed to measure the activity of the chimeric transporters, under specific conditions.

Endocytosis is a critical component of plasma membrane dynamics, by allowing the removal of proteins such as transporters or receptors in response to environmental cues. In yeast, transporter endocytosis requires their ubiquitylation at the plasma membrane by the Nedd4-like E3 ubiquitin ligase, Rsp5. Since the ubiquitylation of a given transporter occurs only in response to specific signals, this raises the question of how substrate specificity is achieved, and how it is regulated dynamically. Various "adaptor" proteins were identified, which may promote the interaction between Rsp5 and its substrates, and may provide a basis for this regulation. However, how they modulate Rsp5 function in response to extracellular stimuli is unknown. We addressed this question by studying a model transporter, Jen1, which is a lactate transporter induced in the presence of lactate and endocytosed in response to glucose (Paiva et al., JBC 2009). We identified an Rsp5 adaptor protein that belongs to the alpha-arrestin family, Art4 (also named Rod1), as essential for Jen1 ubiquitylation and endocytosis of in response to glucose. Interestingly, when cells are grown in lactate medium to induce Jen1 expression, Art4 is strongly phosphorylated by the yeast AMPK homologue, Snf1. Addition of glucose, known to trigger Jen1 endocytosis, leads to a rapid dephosphorylation of Art4, a process that requires the PP1 phosphatase regulatory subunit Reg1. This dephosphorylation allows Art4 ubiquitylation by Rsp5, and we provide details on the molecular mechanism of this regulation. We also show that Art4 ubiquitylation is required for Jen1 endocytosis. Therefore, a switch in Art4 post-translational modifications occurs in response to glucose and is required to modulate its function as an adaptor of Rsp5. This establishes yeast arrestin-like proteins as key regulators of transporter endocytosis in response to extracellular signals.

Carboxylic acids are widely used in pharmaceutical and food industries and new industrial uses are continually emerging. The first step of carboxylic acids metabolism is their entrance to the cell and understanding in detail this process has significant biotechnological relevance. The first carboxylic acids permease found in Saccharomyces cerevisiae was a lactate/H+ symporter coded by JEN1 gene. In Kluyveromyces lactis, as well as in Candida albicans, two genes were described that encode two JEN1-like transporters specific for mono-e and dicarboxylate uptake, KlJEN1/KlJEN2, and CaJEN1/CaJEN2 respectively1,2,3. The sequencing of other hemiascomycetes genomes by the Genolevures project has demonstrated the existence of a family of Jen1p homologs in other different yeasts. Some of these yeasts have more than two homologues, but its function is still unclear. This is the case of Yarrowia lipolytica, a yeast species able to use a broad range of substrates and that presents 6 homologues to Jen1p. A mediated transport system for mono-, di- and tricarboxylates was found in this yeast, suggesting that the respective gene family can putatively code for carboxylate permeases. In order to assess their functional characterization, these genes have been heterologously expressed in the strain S. cerevisiae jen1∆ady2∆ that presents no activity for a carboxylate permease4. All the transformant strains showed a slightly improved growth in mono- and di- but not tricarboxylic acids containing media. Alongside, RT-PCR assays showed that YLJEN expression is induced by carboxylic acids and YlJen-GFP chimeric proteins were all located in the plasma membrane. Taken together, these results suggest that these Jen1 homologues can mediate the transport of carboxylic acids in Y. lipolytica., This work was supported by the project CESPU 02-GBMC-CICS-11.

International audience; Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that Casein Kinase 1-dependent phosphorylation, and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-K63 linked chain(s), and Jen1-Lys338 is one of the target residues. Ubiquitin-K63 linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies (MVBs). Jen1 is one of the few examples for which ubiquitin-K63 linked chain(s) were shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at MVBs.

Poster apresentado no "Simpósio Corantes e Pigmentos Orgânicos", na Universidade de Trás-os-Montes e Alto Douro, em Vila Real, Portugal, em Novembro de 2004., Our group is undertaking an investigation on the potential application of ascomycete yeasts to the decolourisation of azo dyes. Two of these strains, Candida zeylanoides (UM2) and Issatchenkia occidentalis (UM41), were isolated from contaminated soil and have been shown to mediate dye decolourisation through reductive cleavage of the azo bond. The rates of colour loss in the presence of yeast cells are independent of their previous exposure to the dye, suggesting that the decolourising activity, under the conditions tested, is constitutive. The process requires intact cells and an external carbon and energy source and depends on pH, temperature and dissolved oxygen. Interestingly, anaerobic conditions do not allow decolourisation. The kinetic study of the cells decolourising activity demonstrated that such activity has a maximum in the late exponential growth phase. Although glucose is the standard carbon and energy source we have also observed decolourisation by cells growing at the expense of ethanol. Decolourisation rates are considerably dependent on the dye structure. Of considerable practical interest is the observation that some of the amines produced by azo dye reduction can be used as carbon and nitrogen sources by the yeast. In order to get further insight on the yeast decolourising activity we have prepared some mutants of a laboratory strain of Saccharomyces cerevisiae and performed inhibition studies. The experimental evidence suggests that major part of the decolourising activity of intact yeast cells is due to a very well characterized plasma membrane redox system., BIOEFTEX Project.

Poster apresentado no International Congress on Yeasts (ICY) -Yeasts in Science and Biotechnology : The Quest for Sustainable Development", 11, Rio de Janeiro, Brasil, Agosto 2004., Azo dyes are synthetic organic colorants which are extensively used in textile, food and cosmetic industries. A large fraction of these dyes is released into the environment even after conventional wastewater treatments. This is a worldwide problem and particularly a problem in regions where textile industries release large quantities of coloured wastewater to water courses. In an attempt to develop a biological treatment for colour removal we isolated several ascomycete yeast strains from contaminated soil based on its capacity to decolourize soluble azo dyes. We studied the process in batch cultures and have demonstrated that the colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines [1]. In order to understand the process involved in this reduction we selected a Saccharomyces cerevisiae strain (BLC0276) with this capacity. This strain reduced model azo dyes in 8-16 h. We decided to further investigate which enzyme(s) could be involved in this reduction. It was found that in S. cerevisiae there is a membrane redox system with an externally directed reductase – the ferric reductase system, responsible for the extracellular reduction of ferric to ferrous iron previous to uptake, and involving the genes fre1 and fre2. Several know inhibitors of this system were investigated like excess of iron in the medium, CCCP, DPI, chloroquine and p-chloromercurybenzoic acid. Several of these compounds managed to inhibit the decolourizing activity. We also deleted the genes fre1 and fre2 in our strain and analysed the effect on decolourizing activity. Once again the process was retarded in fre1 and fre1fre2 strains. The effect of fre2 deletion was negligible. The deletion of fre1 retarded the decolourizing capability showing the involvement of this system. However the capacity was not completely removed indicating that cells have an alternative reducing system. [1] Ramalho, P.A., Scholze, H., Cardoso, M.H., Ramalho, M.T., Oliveira-Campos, A.M. 2002 Improved conditions for the aerobic reductive decolourization of azo dyes by Candida zeylanoides. Enz Microbiol Technol 31: 848-854., BIOEFTEX Project.

Poster apresentado em 2004 Yeast Genetics and Molecular Biology Meeting, Seattle, USA, 27 Jul. - 1 Ag. 2004, Portuguese Government - Ph.D. Grant from the (BD/1898/98), FEBS - short term fellowship, Portuguese Government - Technichal Grant POCTI/BME/36625/2000 , POCTI/1999/36625

Apresentação efectuada nas Jornadas de Biologia de Leveduras “Professor Nicolau van Uden”, 12, Universidade de Aveiro, Portugal, 13-15 Maio 2004., In an attempt to develop a biological treatment for colour removal we isolated several ascomycete yeast strains from contaminated soil based on its capacity to decolourize soluble azo dyes. We studied the process in batch cultures and have demonstrated that the colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines [1]. To understand the process behind the azo decolourising capacity of some yeast strains isolated by our group [1,2] we selected a Saccharomyces cerevisiae strain (BLC0276) with this capacity. This strain was able to decolourize the media in 8 to 16 h. With evidences from a previous work and with the knowledge that the plasma membrane redox systems are known to be ubiquitous and multifunctional, we decided to explore the involvement of one of these systems on this feature of S.cerevisiae – the ferric reductase system. This system is responsible for the reduction of ferric to ferrous iron previous to its uptake by the yeast cell. First we compared both ferric and azo reducing activities along the growth phases. Both have an activity peak in the late exponential growth phase. These peaks correspond in growing cultures to the phase where the dye is more actively removed. Then several known inhibitors were added to the media, like carbonylcyanide m-chlorophenylhydrazone (CCCP), diphenylene iodonium (DPI), p-chloromercuribenzoate (pCMB) and chloroquine (CQ), and different levels of inhibition on both ferric and azo reductase activities were observed. The genes Fre1 and Fre2 were deleted and the effect on the decolourising activity was analysed in the mutant strains. Once again the process was retarded in fre1 and fre1fre2 strains. The effect of Fre2 deletion was negligible. The deletion of Fre1 retarded the decolourising capability showing the involvement of this system in the azo decolorizing capacity of S.cerevisiae. However the capacity was not completely removed indicating that cells have an alternative reducing system. [1] Ramalho, P.A., Scholze, H., Cardoso, M.H., Ramalho, M.T., Oliveira-Campos, A.M. 2002 Improved conditions for the aerobic reductive decolourization of azo dyes by Candida zeylanoides. Enz Microbiol Technol 31: 848-854. [2] Ramalho, P.A., Cardoso, M.H., Cavaco-Paulo, A., Ramalho, M.T. 2004 Characterisation of the azo reductase activity in a novel ascomycete yeast strain. Accepted for publication in Appl Environ Microbiol 70, BIOEFTEX Project.

Poster apresentado na International Conference on Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, 7, Asilomar, Califórnia, Estados Unidos da América, 14-18 Abril 2004., Unspecific bacterial reduction of azo dyes is a widely studied process in correlation with the biological treatment of coloured waste waters but the enzyme system associated to this bacterial capability has never been positively identified. Several ascomycete yeast strains, isolated by our group from contaminated soils, display similar decolourising activities. A study of the yeast-mediated process in batch culture demonstrated that colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines. In order to get a better understanding of the azoreductase activity of yeast cells we selected a Saccharomyces cerevisiae strain (BLC0276) with a high decolourising capacity. A likely candidate might be the plasma membrane ferric reductase system. Both azoreductase and ferric reductase have peak activities in the late exponential growth phase, and their substrates -ferricyanide and soluble azo dyes - are impermeant to the cell plasma membrane. This hypothesis was confirmed through two different lines of evidence: the use of different inhibitors and the construction of mutants defective in the iron reductase system. Inhibitors like carbonylcyanide m-chlorophenylhydrazone (CCCP), diphenylene iodonium (DPI), p-chloromercuribenzoate (pCMB) and chloroquine (CQ), were tested. In most cases the percentage inhibition of both activities was similar. The genes Fre1 and Fre2 were deleted and the effect on the decolourising activity was analysed in the mutant strains. The effect of Fre2 deletion was negligible, but fre1 and fre1fre2 strains showed a much reduced decolourising capability. These results demonstrate the involvement of the ferric reductase system in the azo decolourising capacity of S. cerevisiae. However the capacity was not completely removed indicating that cells have an alternative reducing system., BIOEFTEX Project.

Poster apresentado em XXI International Conference on Yeast Genetics and Molecular Biology, Gotemburgo, Suécia, 7 Jul. - 12 Jul. 2003

Green Fluorescent protein (GFP) from Aequorea ictoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalyticcentre activity of 123 s-1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p-GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Ddoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease., FEBS - short-term fellowship., Portugal - Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) - POCTI/1999/BME/36 625., Portuguese Government - PRAXIS XXI-BD/1898/98.

We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins. We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S. cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane. Ayeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources. Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wildtype strain. Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate. However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter. The mch1–5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism. Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes. However, pyruvate uptake into isolated mitochondria was not affected in the mch1–5 mutant strain. It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts., União Europeia (UE) – grant BIO4-CT97-2294 (EUROFAN2)., Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI-BD/1898/98.