Physical, genetic and functional interactions between the eisosome protein Pil1 and the MBOAT O-acyltransferase Gup1.

The Saccharomyces cerevisiae MBOAT O-acyltransferase Gup1 is involved in many processes, including cell wall and membrane composition and integrity, and acetic acid-induced cell death. Gup1 was previously shown to interact physically with the mitochondrial membrane VDAC (Voltage-Dependent Anion Channel) protein Por1 and the ammonium transceptor Mep2. By co-immunoprecipitation, the eisosome core component Pil1 was identified as a novel physical interaction partner of Gup1. The expression of PIL1 and Pil1 protein levels were found to be unaffected by GUP1 deletion. In ∆gup1 cells, Pil1 was distributed in dots (likely representing eisosomes) in the membrane, identically to wt cells. However, ∆gup1 cells presented 50% less Pil1-GFP dots/eisosomes, suggesting that Gup1 is important for eisosome formation. The two proteins also interact genetically in the maintenance of cell wall integrity, and during arsenite and acetic acid exposure. We show that Δgup1 Δpil1 cells take up more arsenite than wt and are extremely sensitive to arsenite and to acetic acid treatments. The latter causes a severe apoptotic wt-like cell death phenotype, epistatically reverting the ∆gup1 necrotic type of death. Gup1 and Pil1 are thus physically, genetically and functionally connected.

Saccharomyces cerevisiae mitochondrial Por1/yVDAC1 (voltage-dependent anion channel 1) interacts physically with the MBOAT O-acyltransferase Gup1/HHATL in the control of cell wall integrity and programmed cell death.

Gup1 is the yeast counterpart of the high eukaryotes HHATL. This and the close homologue Gup2/HHAT regulate the Hedgehog morphogenic, developmental pathway. In yeasts, a similar paracrine pathway is not known though the Δgup1 mutant is associated with morphology and proliferation/death processes. As a first step toward identifying the actual molecular/enzymatic function of Gup1, this work identified by co-immunoprecipitation the yeast mitochondria membrane VDAC1/Por1 as a physical partner of Gup1. Gup1 locates in the ER and the plasma membrane. It was now confirmed to further locate, as Por1, in the mitochondrial sub-cellular fraction. The yeast Por1-Gup1 association was found important for (i) the sensitivity to cell wall perturbing agents and high temperature, (ii) the differentiation into structured colonies, (iii) the size achieved by multicellular aggregates/mats and (iv) acetic-acid-induced Programmed Cell Death. Moreover, the absence of Gup1 increased the levels of POR1 mRNA, while decreasing the amounts of intracellular Por1, which was concomitantly previously known to be secreted by the mutant but not by wt. Additionally, Por1 patchy distribution in the mitochondrial membrane was evened. Results suggest that Por1 and Gup1 collaborate in the control of colony morphology and mat development, but more importantly of cellular integrity and death.

Yeast Gup1(2) Proteins Are Homologues of the Hedgehog Morphogens Acyltransferases HHAT(L): Facts and Implications.

In multiple tissues, the Hedgehog secreted morphogen activates in the receiving cells a pathway involved in cell fate, proliferation and differentiation in the receiving cells. This pathway is particularly important during embryogenesis. The protein HHAT (Hedgehog O-acyltransferase) modifies Hh morphogens prior to their secretion, while HHATL (Hh O-acyltransferase-like) negatively regulates the pathway. HHAT and HHATL are homologous to Saccharomyces cerevisiae Gup2 and Gup1, respectively. In yeast, Gup1 is associated with a high number and diversity of biological functions, namely polarity establishment, secretory/endocytic pathway functionality, vacuole morphology and wall and membrane composition, structure and maintenance. Phenotypes underlying death, morphogenesis and differentiation are also included. Paracrine signalling, like the one promoted by the Hh pathway, has not been shown to occur in microbial communities, despite the fact that large aggregates of cells like biofilms or colonies behave as proto-tissues. Instead, these have been suggested to sense the population density through the secretion of quorum-sensing chemicals. This review focuses on Gup1/HHATL and Gup2/HHAT proteins. We review the functions and physiology associated with these proteins in yeasts and higher eukaryotes. We suggest standardisation of the presently chaotic Gup-related nomenclature, which includes KIAA117, c3orf3, RASP, Skinny, Sightless and Central Missing, in order to avoid the disclosure of otherwise unnoticed information.

XYLH encodes a xylose/H+ symporter from the highly related yeast species Debaryomyces fabryi and Debaryomyces hansenii.

The closely related yeasts Debaryomyces fabryi and Debaryomyces hansenii are excellent xylose consumers. We previously described the activity of a high-affinity xylose/H(+) symport from an industrial strain of D. hansenii subsequently reclassified as D. fabryi. We now report the identification of the gene encoding this permease, AY347871.2. This was retrieved from D. fabryi gDNA using a degenerate primer PCR strategy, based on conserved regions from the amino acid sequences of three well-characterized bacterial xylose/H(+) symporters. This sequence is 86% identical to another, DEHA2C11374p from D. hansenii type strain. DEHA2C11374p was conceptually ascribed to the major facilitator superfamily. The putative amino acid sequence of AY347871.2 and DEHA2C11374p presented a hydrophobicity pattern compatible with plasma membrane proteins. The last was functionally expressed in Saccharomyces cerevisiae. The sensitivity of transport activity to a protonophore confirmed its dependence on proton motive force, as expected from a symporter. We named D. fabryi AY347871.2 and D. hansenii DEHA2C11374p as XYLH from Xylose/H(+) symport. Based on the very high similarity, we suggested that Scheffersomyces stipitis Xut3 and Aspergillus nidulans AN8400.2 may also encode xylose high-affinity permeases.

Programmed cell death in Saccharomyces cerevisiae is hampered by the deletion of GUP1 gene.

BACKGROUND: During the past years, yeast has been successfully established as a model to study mechanisms of programmed cell death regulation. Saccharomyces cerevisiae commits to cell death showing typical hallmarks of metazoan apoptosis, in response to different stimuli. Gup1p, an O-acyltransferase, is required for several cellular processes that are related to apoptosis development, such as rafts integrity and stability, lipid metabolism including GPI anchor correct remodeling, proper mitochondrial and vacuole function, bud site selection and actin dynamics. Therefore, we hypothesize that apoptotic process would be affected by GUP1 deletion. RESULTS: In the present work we used two known apoptosis inducing conditions, chronological aging and acetic acid, to assess several apoptotic markers in gup1∆ mutant strain. We found that this mutant presents a significantly reduced chronological lifespan as compared to Wt and it is also highly sensitive to acetic acid treatment. In addition, it presents extremely high levels of ROS. There were notorious differences on apoptotic markers between Wt and gup1∆ mutant strains, namely on the maintenance of plasma membrane integrity, on the phosphatidylserine externalization, on the depolarization of mitochondrial membrane and on the chromatin condensation. Those suggested that the mutant, under either condition, probably dies of necrosis and not from apoptosis. CONCLUSIONS: To Gup1p has been assigned an important function on lipid rafts assembly/integrity, lipid metabolism and GPI anchor remodeling. Our results provide, for the first time, the connection of the integrity of yeast lipid rafts and apoptosis induction and/or signaling, giving new insights into the molecular mechanisms underlying this process in yeast.

Saccharomyces cerevisiae glycerol/H+ symporter Stl1p is essential for cold/near-freeze and freeze stress adaptation. A simple recipe with high biotechnological potential is given.

BACKGROUND: Freezing is an increasingly important means of preservation and storage of microbial strains used for many types of industrial applications including food processing. However, the yeast mechanisms of tolerance and sensitivity to freeze or near-freeze stress are still poorly understood. More knowledge on this regard would improve their biotechnological potential. Glycerol, in particular intracellular glycerol, has been assigned as a cryoprotectant, also important for cold/near-freeze stress adaptation. The S. cerevisiae glycerol active transporter Stl1p plays an important role on the fast accumulation of glycerol. This gene is expressed under gluconeogenic conditions, under osmotic shock and stress, as well as under high temperatures. RESULTS: We found that cells grown on STL1 induction medium (YPGE) and subjected to cold/near-freeze stress, displayed an extremely high expression of this gene, also visible at glycerol/H+ symporter activity level. Under the same conditions, the strains harbouring this transporter accumulated more than 400 mM glycerol, whereas the glycerol/H+ symporter mutant presented less than 1 mM. Consistently, the strains able to accumulate glycerol survive 25-50% more than the stl1Δ mutant. CONCLUSIONS: In this work, we report the contribution of the glycerol/H+ symporter Stl1p for the accumulation and maintenance of glycerol intracellular levels, and consequently cell survival at cold/near-freeze and freeze temperatures. These findings have a high biotechnological impact, as they show that any S. cerevisiae strain 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.