| 1. |
- Molin, Mikael, 1973, et al.
(författare)
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Protein kinase A controls yeast growth in visible light
- 2020
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400–700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.
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| 2. |
- Forsmark, Annabelle, 1973, et al.
(författare)
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Quantitative Proteomics of Yeast Post-Golgi Vesicles Reveals a Discriminating Role for Sro7p in Protein Secretion
- 2011
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Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 12:6, s. 740-753
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Tidskriftsartikel (refereegranskat)abstract
- We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.
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| 3. |
- Kohler, A., et al.
(författare)
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High-throughput biochemical fingerprinting of Saccharomyces cerevisiae by Fourier transform infrared spectroscopy
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:2
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Tidskriftsartikel (refereegranskat)abstract
- Single-channel optical density measurements of population growth are the dominant large scale phenotyping methodology for bridging the gene-function gap in yeast. However, a substantial amount of the genetic variation induced by single allele, single gene or double gene knock-out technologies fail to manifest in detectable growth phenotypes under conditions readily testable in the laboratory. Thus, new high-throughput phenotyping technologies capable of providing information about molecular level consequences of genetic variation are sorely needed. Here we report a protocol for high-throughput Fourier transform infrared spectroscopy (FTIR) measuring biochemical fingerprints of yeast strains. It includes high-throughput cultivation for FTIR spectroscopy, FTIR measurements and spectral pretreatment to increase measurement accuracy.We demonstrate its capacity to distinguish not only yeast genera, species and populations, but also strains that differ only by a single gene, its excellent signal-to-noise ratio and its relative robustness to measurement bias. Finally, we illustrated its applicability by determining the FTIR signatures of all viable Saccharomyces cerevisiae single gene knock-outs corresponding to lipid biosynthesis genes. Many of the examined knock-out strains showed distinct, highly reproducible FTIR phenotypes despite having no detectable growth phenotype. These phenotypes were confirmed by conventional lipid analysis and could be linked to specific changes in lipid composition. We conclude that the introduced protocol is robust to noise and bias, possible to apply on a very large scale, and capable of generating biologically meaningful biochemical fingerprints that are strain specific, even when strains lack detectable growth phenotypes. Thus, it has a substantial potential for application in the molecular functionalization of the yeast genome.
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| 4. |
- Warringer, Jonas, 1973, et al.
(författare)
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Trait variation in yeast is defined by population history.
- 2011
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Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- A fundamental goal in biology is to achieve a mechanistic understanding of how and to what extent ecological variation imposes selection for distinct traits and favors the fixation of specific genetic variants. Key to such an understanding is the detailed mapping of the natural genomic and phenomic space and a bridging of the gap that separates these worlds. Here we chart a high-resolution map of natural trait variation in one of the most important genetic model organisms, the budding yeast Saccharomyces cerevisiae, and its closest wild relatives and trace the genetic basis and timing of major phenotype changing events in its recent history. We show that natural trait variation in S. cerevisiae exceeds that of its relatives, despite limited genetic variation, and follows the population history rather than the source environment. In particular, the West African population is phenotypically unique, with an extreme abundance of low-performance alleles, notably a premature translational termination signal in GAL3 that cause inability to utilize galactose. Our observations suggest that many S. cerevisiae traits may be the consequence of genetic drift rather than selection, in line with the assumption that natural yeast lineages are remnants of recent population bottlenecks. Disconcertingly, the universal type strain S288C was found to be highly atypical, highlighting the danger of extrapolating gene-trait connections obtained in mosaic, lab-domesticated lineages to the species as a whole. Overall, this study represents a step towards an in-depth understanding of the causal relationship between co-variation in ecology, selection pressure, natural traits, molecular mechanism, and alleles in a key model organism.
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| 5. |
- Forsmark, Annabelle, 1973
(författare)
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Functional Characterization of the Yeast Tumour Suppressor Homologue Sro7p
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Correct targeting of newly synthesized proteins to appropriate domains of the cell membrane is crucial to cellular architecture, polarity and function, making it no surprise that many proteins of the secretory machinery are conserved throughout evolution. This work presents a functional characterisation of the Saccharomyces cerevisiae cell polarity protein and tumour suppressor homologue, Sro7p. This protein, and its paralogue Sro77p, belong to the Lgl-family of WD-40 repeat proteins that are conserved from yeast to human. Deletion of Lgl genes produces different phenotypes that all seem to share the common denominator of defective targeting of critical cell surface proteins. Yeast cells lacking SRO7 become sensitive to NaCl and we here show that this defect is due to mis-targeting of the sodium transporter Ena1p. In sro7 mutants Ena1p becomes routed to the vacuole for degradation via the multi-vesicular body (MVB) pathway, instead of being properly expressed at the cell surface. Isolation and analysis of post-Golgi secretory vesicles showed a defective sorting of Ena1p into these vesicles from sro7 mutants, implying mis-sorting in late Golgi or early endosomes. The diversion of Ena1p into the MVB pathway further required ubiquitylation by the ubiquitin ligase Rsp5p. Isolation of suppressors of the sro7 salt sensitivity identified two genes of unknown function, RSN1 encoding a trans-membrane protein, and ART5 (RSN2), encoding an arrestin-like protein. Deletion of either gene in sro7 mutants re-establishes salt tolerance and retargets Ena1p to the cell surface. Previous proteomic studies have shown that Art5p interacts with Rsp5p and we showed that deletion of ART5 in sro7 mutants inhibits ubiquitylation of Ena1p. Our data are consistent with Art5p being a selective adaptor protein that helps Rsp5p recruiting Ena1p for ubiquitylation. To identify further candidate proteins for mis-sorting in salt stressed sro7 mutants we performed the first proteomic analysis of purified yeast post-Golgi vesicles (PGVs), using quantitative proteomics techniques. By this analysis we could identify 107 genuine vesicle residents in control yeast cells, including a number of cargo proteins not previously identified in PGVs. Vesicles derived from sro7 mutants contained essentially the same list of proteins but were depleted of a subset of proteins, thus being candidates for mis-routing. The present study finally analysed possible Lgl conservation in plants by characterising two Arabidopsis thaliana Lgl homologues. Sequence based modelling showed that both proteins can fold into the twin β-propellers shown by the published Sro7p crystal structure. However, only one of the proteins, AtLGL1, could partially substitute for the yeast Sro7/77 proteins. The other, AtLGL2 showed structural similarities with tomosyn that is known to regulate vesicle fusion in mammals. Homozygous T-DNA insertion mutants in A. thaliana exhibited defects in lateral root formation, a phenotype associated with changed cell- and tissue polarity.
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| 6. |
- Granath, Katarina, 1971, et al.
(författare)
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The YIGI (YPL201c) encoded protein is involved in regulating anaerobic glycerol metabolism in Saccharomyces cerevisiae
- 2005
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 22:16, s. 1257-1268
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Tidskriftsartikel (refereegranskat)abstract
- Under anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD+ from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (YpI201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two-hybrid tests with the GPP1 and GPP2 encoded glycerol 3-phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post-transcriptional levels. In agreement with such a role, a GFP-tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level. Copyright (c) 2005 John Wiley & Sons, Ltd.
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| 7. |
- Wadskog, Ingrid, et al.
(författare)
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The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface.
- 2006
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Ingår i: Molecular biology of the cell. - 1059-1524 .- 1939-4586. ; 17:12, s. 4988-5003
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Tidskriftsartikel (refereegranskat)abstract
- The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.
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