Haeuptle MA, Pujol FM, Neupert C, Winchester B, Kastaniotis AJ, Aebi M, Hennet T
Am J Hum Genet. 2008 Mar;82(3):600–6
N-linked glycosylation is an essential posttranslational modification of proteins in eukaryotes. The substrate of N-linked glycosylation, dolichol pyrophosphate (DolPP)-GlcNAc(2)Man(9)Glc(3), is assembled through a complex series of ordered reactions requiring the translocation of the intermediate DolPP-GlcNAc(2)Man(5) structure across the endoplasmic-reticulum membrane. A young patient diagnosed with a congenital disorder ofglycosylation characterized by an intracellular accumulation of DolPP-GlcNAc(2)Man(5) was found to carry a homozygous point mutation in the RFT1gene. The c.199C-->T mutation introduced the amino acid substitution p.R67C. The human RFT1 protein shares 22% identity with its yeast ortholog, which is involved in the translocation of DolPP-GlcNAc(2)Man(5) from the cytosolic into the lumenal side of the endoplasmic reticulum. Despite the low sequence similarity between the yeast and the human RFT1 proteins, we demonstrated both their functional orthology and the pathologic effect of the human p.R67C mutation by complementation assay in Deltarft1 yeast cells. The causality of the RFT1 p.R67C mutation was further established by restoration of normal glycosylation profiles in patient-derived fibroblasts after lentiviral expression of a normal RFT1 cDNA. The definition of the RFT1 defect establishes the functional conservation of the DolPP-GlcNAc(2)Man(5) translocation process in eukaryotes. RFT1deficiency in both yeast and human cells leads to the accumulation of incomplete DolPP-GlcNAc(2)Man(5) and to a profound glycosylation disorderin humans.
Smaczynska-de Rooij II, Costa R, Ayscough KR
Traffic. 2008 Apr;9(4):559–73
Phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] is a key regulator of endocytosis. PtdIns(4,5)P2 generation at the plasma membrane in yeastis mediated by the kinase Mss4p, but the mechanism underlying the temporal and spatial activation of Mss4p to increase formation of PtdIns(4,5)P2at appropriate sites is not known. Here, we show that ADP ribosylation factor (Arf)3p, the yeast homologue of mammalian Arf6, is necessary for wild-type levels of PtdIns(4,5)P2 at the plasma membrane. Arf3p localizes to dynamic spots at the membrane, and the behaviour of these is consistent with it functioning in concert with endocytic machinery. Localization of Arf3p is disrupted by deletion of genes encoding an ArfGAP homology protein Gts1p and a guanine nucleotide exchange factor Yel1p. Significantly, deletion of arf3 causes a reduction in PtdIns(4,5)P2 at the plasma membrane, while increased levels of active Arf3p, caused by deletion of the GTPase-activating protein Gts1, increase PtdIns(4,5)P2 levels. Furthermore, elevated Arf3p correlates with an increase in the number of endocytic sites. Our data provide evidence for a mechanism in yeast to positively regulate plasma membrane production of PtdIns(4,5)P2 levels and that these changes impact on endocytosis.
Song W-Q, Qin Y-M, Saito M, Shirai T, Pujol FM, Kastaniotis AJ, Hiltunen JK, Zhu YX
J Exp Bot. 2009;60(6):1839–48
Very long chain fatty acids are important components of plant lipids, suberins, and cuticular waxes. Trans-2-enoyl-CoA reductase (ECR) catalyses the fourth reaction of fatty acid elongation, which is NADPH dependent. In the present study, the expression of two cotton ECR (GhECR) genes revealed by quantitative RT-PCR analysis was up-regulated during cotton fibre elongation. GhECR1 and 2 each contain open reading frames of 933 bp in length, both encoding proteins consisting of 310 amino acid residues. GhECRs show 32% identity to Saccharomyces cerevisiae Tsc13p at the deduced amino acid level, and the GhECR genes were able to restore the viability of the S. cerevisiae haploid tsc13-deletion strain. A putative non-classical NADPH-binding site in GhECR was predicted by an empirical approach. Site-directed mutagenesis in combination with gas chromatography-mass spectrometry analysis suggests that G(5X)IPXG presents a putative novel NADPH-binding motif of the plant ECR family. The data suggest that both GhECR genes encode functional enzymes harbouring non-classical NADPH-binding sites at their C-termini, and are involved in fatty acid elongation during cotton fibre development.
Albertin W, Marullo P, Aigle M, Bourgais A, Bely M, Dillmann C, DE Vienne D, Sicard D
J Evol Biol. 2009 Nov;22(11):2157–70
Partial or whole-genome duplications have played a major role in the evolution of new species. We have investigated the variation of ploidy level in a panel of domesticated strains of Saccharomyces cerevisiae coming from different geographical origins. Segregation studies and crosses with tester strains of different ploidy levels showed that part of the strains were well-balanced autotetraploids displaying tetrasomic inheritance. The presence of up to four different alleles for various loci is consistent with a polyploidization mechanism relying on the fusion of two nonreduced meiospores coming from two S. cerevisiae strains. Autotetraploidy was also in accordance with karyotype and flow cytometry analyses. Interestingly, most bakery strains were tetraploids, suggesting a link between ploidy level and human use. The null or drastically reduced fertility of the hybrids between tetraploid and diploid strains indicated that domesticated S. cerevisiae strains are composed of two groups isolated by post-zygotic reproductivebarriers.
Marullo P, Mansour C, Dufour M, Albertin W, Sicard D, Bely M, Dubourdieu D
FEMS Yeast Res. 2009 Dec;9(8):1148–60
During red wine fermentation, high temperatures may cause stuck fermentation by affecting the physiology of fermenting yeast. This deleterious effect is the result of the complex interaction of temperature with other physicochemical parameters of grape juice, such as sugar and lipid content. The genetic background of fermenting yeast also interacts with this complex matrix and some strains are more resistant to high temperatures than others. Here, the temperature tolerance of nine commercial starters was evaluated, demonstrating that, at high sugar concentrations, half of them are sensitive to temperature. Using a classical backcross approach, one thermo-sensitive commercial starter was genetically improved by introducing quantitative trait loci conferring resistance to temperature. With this breeding program it is possible to obtain a thermo-resistant strain sharing most of its genome with the initial commercial starter. The parental and improved strains were compared for population growth and fermentation ability in various conditions. Despite their common genetic background, these two strains showed slight physiological differences in response to environmental changes that enable identification of the key physiological parameters influencing stuck fermentation.
Masneuf-Pomarède I, Bely M, Marullo P, Lonvaud-Funel A, Dubourdieu D
Int J Food Microbiol. 2010 Apr 30;139(1-2):79–86
Among Saccharomyces yeast, S. cerevisiae and S. bayanus var. uvarum are related species, sharing the same ecosystem in sympatry. The physiological and technological properties of a large collection of genetically-identified S. bayanus var. uvarum wine strains were investigated in a biometric study and their fermentation behavior was compared at 24 degrees C and 13 degrees C. The variability of the phenotypic traits was considered at both intraspecific and interspecific levels. Low ethanol tolerance at 24 degrees C and production of high levels of 2-phenylethanol and its acetate were clearly revealed as discriminative technological traits, distinguishing the S. bayanus var. uvarum strains from S. cerevisiae. Although some S. bayanus var. uvarum strains produced very small amounts of acetic acid, this was not a species-specific trait, as the distribution of values was similar in both species. Fermentation kinetics at 24 degrees C showed that S. bayanus var. uvarum maintained a high fermentation rate after Vmax, with low nitrogen requirements, but stuck fermentations were observed at later stages. In contrast, a shorter lag phase compared with S.cerevisiae, higher cell viability, and the ability to complete alcoholic fermentation at 13 degrees C confirmed the low-temperature adaptation trait of S.bayanus var. uvarum. This study produced a phenotypic characterization data set for a collection of S. bayanus var. uvarum strains, thus paving the way for industrial developments using this species as a new genetic resource.
Naumov GI, Naumova ES, Masneuf-Pomarède I
Antonie Van Leeuwenhoek. 2010 Jun;98(1):1–7
Direct genetic testing for hybrid sterility unambiguously showed that the newly described yeast Saccharomyces arboricolus Wang et Bai is reproductively isolated from Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces kudriavzevii,Saccharomyces mikatae and Saccharomyces paradoxus and, therefore, represents a new biological species of the genus Saccharomyces. Combined phylogenetic analysis of the rDNA repeat sequences (18S, 26S, ITS), nuclear ACT1 and mitochondrial ATP9 genes revealed that S.arboricolus, along with S. kudriavzevii and S. bayanus, is distantly related to the other four biological species.
Lopes CA, Barrio E, Querol A
FEMS Yeast Res. 2010 Jun;10(4):412–21
Saccharomyces kudriavzevii, a yeast species described from a pair of strains isolated from decayed leaves in Japan, has recently been isolated from oak barks in Portugal. Some data suggest that these European S. kudriavzevii populations could be closely related to the S. kudriavzevii genetic background present in natural hybrids isolated from wines and beers in different regions of Europe. However, a more exhaustive study of European S. kudriavzevii natural populations is necessary to confirm this observation. In this study, new S. kudriavzevii isolates were recovered from oak trees in different areas in Spain, and identified and characterized according to their molecular and physiological features. The restriction analysis of 35 nuclear genes amplified by PCR demonstrated that all Spanish S. kudriavzevii isolates exhibit three gene alleles different from those present in the Japanese S. kudriavzevii type strain, but identical to the alleles previously reported for natural S. cerevisiaexS. kudriavzevii hybrids. These results clearly support the hypothesis of a European origin for these natural hybrids.
Pang Y, Wang H, Song W-Q, Zhu Y-X
Plant Biol (Stuttg). 2010 Nov;12(6):903–9
The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthaseactivity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors ofATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation.
Naumova ES, Naumov GI, Michailova YV, Martynenko NN, Masneuf-Pomarède I
Res Microbiol. 2011 Feb;162(2):204–13
Intraspecies polymorphism of the yeast Saccharomyces bayanus var. uvarum was studied using the polymerase chain reaction with a microsatellite primer (GTG)(5). Sixty-nine strains of different origins were analyzed. There existed a correlation between PCR patterns of the strains and the source of their isolation: the type of wine and the particular winemaking region. Southern hybridization analysis revealed for the first time introgression between Saccharomyces cerevisiae and S. bayanus var. uvarum. Two strains isolated from alcoholic beverages in Hungary and identified by genetic analysis as S. bayanus var. uvarum were found to harbor a number of S. cerevisiae subtelomeric sequences: Y', SUC, RTM and MAL.