Centro de Biologia Molecular e Ambiental

Centre of Molecular and Environmental Biology

Yeast: signalling, walls and membranes

Research highlights

This line of research, dedicated to the cell surface processes, includes studies in plasma membrane transporters involved in uptake, compartmentalization and mobilization of specific compounds, their structure and regulation, as well as signaling controlling the wall and membrane structure and cell differentiation. The research around transporters has focused on glycerol and carboxylic acids transporters, essentially in Saccharomyces cerevisiae, but also in other yeast species with diverse relevance in health and biotechnology.

In S. cerevisiae glycerol plays important roles in diverse cellular processes. The modulation of glycerol internal concentrations depends on transport and retention through channel closure besides metabolism. The glycerol H+/symporter Stl1p, identified by our group, is tightly and complexly regulated in response to numerous stimuli, including osmotic stress. We showed that it is also induced by high temperatures, and more importantly that under such conditions glucose repression was alleviated. This is, to our knowledge, the only case where catabolic repression is overcome by heat stress. Recently, we have shown that Stl1p is also highly induced by cold/freeze stress, contributing to the survival of the cell at cold/near-freeze and freeze temperatures. These last findings have a high biotechnological impact, as they show that any S. cerevisiae strains already in use can become more resistant to cold/freeze-thaw stress just by simply adding glycerol to the broth. The combination of low temperatures with extracellular glycerol will induce the transporter Stl1p. This solution avoids the use of transgenic strains, in particular in food industry.

A second highlight concerns the glycerol-related gene GUP1, belonging to the membrane-bound O-acyl transferases family and, also identified by our group. This was shown to interfere in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly.

The number and diversity of cellular processes in which this protein is involved was considerably enlarged. In C. albicans Gup1p was for the first time assessed by our group and it was shown to be involved in the mechanisms underlying drug resistance and importantly to interfere dramatically in dimorphic transition, as well as biofilm formation capabilities (see also the section Yeast: pathogens & their interaction with host cells).

Filipin stained sterols distribution. Sterols distribute in patches at wt plasma membrane level, in clear contrast at Scgup? mutant plasma membrane filipin stained sterols distribute evenly, mirroring the disturbance on the sphingolipid-sterol-ordered domains integrity/assembly

Altogether, these results gave rationale to the hypothesis currently under our study, that Gup1p might govern a morphogenic signalling pathway, identically to what happens with the mammalian Gup1p counterpart, HHATL (Hedgehog O-acyl transferase-like protein).

A third line of research focuses on the physiological and structural/functional analyses, as well as on studies regarding the mechanisms of regulation of carboxylic acids transporters in yeast, namely Jen1-lactate transporter and Ady2p-acetate transporter, which have been identified by our group. This work was developed under the scope of a FCT grant.

In the past years we have identified and characterized homologues to JEN1 of S. cerevisiae in different yeast species namely C. albicans (see also the section Yeast: pathogens & their interaction with host cells) and Kluyveromyces lactis.

Jen1p trafficking and turnover. The lactate permease is tightly regulated upon glucose addition via ubiquitylation and targeting to the endocytic pathway.

These studies allowed the identification and characterization of the Jen2p sub-family of dicardoxylates permeases, shared by malate, succinate and other short-chain dicarboxylates.

Additionally, we studied the trafficking and turnover of Jen1p in S. cerevisiae in detail, particularly the role played by ubiquitylation events at various steps of the endocytic pathway.

We have further demonstrated, for the first time, the key role of ubiquitin-K63 linked chain(s) in the correct trafficking of Jen1 at two stages of endocytosis: endocytic internalization and sorting at MVBs.

Moreover we developed structural/functional studies based on evolutional conservation between Jen1 homologs and/or divergence between Jen1 and Jen2 proteins. This approach was complemented by a Jen1p 3D model obtained in silico by homology threading with the bacterial transporters LacY and GlpT, belonging to the major facilitator superfamily. With this approach we were able to identify new Jen1p functional domains involved in the substrate translocation pathway.

3D predicted structure of Jen1p (viewed from the top). The topology was modelled using the GlpT permease on HHpred Modeller sofware. The figure was prepared with program Swiss PDB viewer.

Ongoing and future works

One main goal in this research line is to acquire in-depth into the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.

For this purpose, a collection of strains expressing the human, mouse and fly GUP1 homologues is under study. This will include the identification of the major intracellular players by transcriptomics and proteomics, as well as the assessment of the yeast extracellular matrix chemical composition.

A second goal aims at characterizing the signalling and trafficking events governing glucose induced down-regulation of Jen1. We will continue to perform the topological analysis of Jen1p using a novel theoretical model created on the basis of Jen1p similarity with GltP permease, and independent substrate docking studies, expecting to reveal a substrate translocation trajectory in the conformation of Jen1p.

In the attempt to obtain a 3D structure of monocarboxylate permeases we will also study the YaaH family of acetate transporters (#2.A.96), which have 6 putative transmembrane domains, with homologues in the 3 domains of life: bacteria, archaea and eukaryota. Using a holistic approach we aim at obtaining physiological, genetic and structural characterization of these proteins.

Overall, these studies, fundamental in nature, will contribute to answer central questions regarding the physiology and biochemistry of permeases in yeast and the mechanisms underlying their regulation, contributing to unravel the complexity of the eukaryotic cell, yeast being the model system.


Key references

Tulha, J., Lima, A., Lucas, C., Ferreira, C. 2010. Saccharomyces cerevisiae glycerol/H+ symporter Stl1p is essential for cold/near-freeze and freeze stress adaptation, Microbial Cell Factories, in press.

Paiva, S., Vieira, N., Nondier, I., Haguenauer-Tsapis, R., Casal, M., Urban-Grimal, D. 2009. Glucose-induced ubiquitylation and endocytosis of the yeast Jen1 transporter: role of k63-linked ubiquitin chains. J Biol Chem. 284: 19228-36.

Casal, M., Paiva, S., Queiros, O., Soares-Silva, I. 2008. Transport of carboxylic acids in yeast. FEMS Microbiol Rev 32: 974-94.

Ferreira, C., Lucas, C. 2008. The yeast O-acyltransferase Gup1p interferes in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly. Biochim. Biophys. Acta 1778: 2648-53.

Ferreira, C., Lucas, C. 2007. Glucose repression over Saccharomyces cerevisiae glycerol/H+ symporter gene STL1 is overcome by high temperatures. FEBS Lett., 581: 1923-1927.

Queiros, O., Pereira, L., Paiva, S., Moradas-Ferreira, P., Casal, M. 2007. Functional analysis of Kluyveromyces lactis carboxylic acids permeases: heterologous expression of KlJEN1 and KlJEN2 genes. Curr Genet 51: 161-169.

Soares-Silva, I., Paiva, S., Diallinas, G., Casal, M. 2007. The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H+ symporter subfamily defines the function of the substrate translocation pathway. Mol Membr Biol, 24: 464-74.

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