A genome-wide resource for high-throughput genomic tagging of yeast ORFs

Meurer, Matthias; Duan, Yuanqiang; Sass, Ehud; Kats, Ilia; Herbst, Konrad; Buchmuller, Benjamin; Dederer, Verena; Huber, Florian; Kirrmaier, Daniel; Štefl, Martin; Laer, Koen Van; Dick, Tobias P.; Lemberg, Marius K.; Khmelinskii, Anton; Levy, Emmanuel D.; Knop, Michael

doi:10.1101/226811

Cited by 4 publications

(8 citation statements)

References 33 publications

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“…Assignment categories were: Bud, Bud Neck, Cell Periphery, Cytosol, ER, Mitochondria, Nuclear Periphery, Nucleolus, Nucleus, Punctate, Vacuole and Vacuole Membrane. (iii) Determination of the SWAT swapping capability (See “Analysis of swapping-procedure efficiency” section); and finally (iv) Sequencing (Anchor-seq) to ensure correct reading frame: We employed a targeted-sequencing strategy detailed in 7 to verify the junction encompassing the 3’ end of the cassette and the 5’ end of each gene. Briefly, we pooled all strains from the SWAT library together, extracted their genomic DNA, sheared it into fragments of 300-800bp that were gel-purified, ligated to Anchor-seq adaptors and submitted to two rounds of PCR.…”

Section: Methodsmentioning

confidence: 99%

“…In a short time period and at a fraction of the cost incurred to date using other approaches, any yeast laboratory can make its very own library harboring a diversity of selection markers, promoters, untranslated regions, targeting signals, fluorophores, affinity tags or any other genetic element of choice. Using this platform, the systematic exploration of any protein is no longer restricted and can be done either with N’ or C’ tagging 7. Together, these approaches should contribute greatly to our knowledge on how the living cell works.…”

Section: Perspectivesmentioning

confidence: 99%

“…Here, we create the first whole-genome SWAT library with an N’ tag (For the complementary C’ SWAT library see 7). Our whole-genome library includes ~90% of yeast genes 8.…”

Section: Introductionmentioning

confidence: 99%

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Genome-wide SWAp-Tag yeast libraries for proteome exploration

et al. 2018

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Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

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Section: Methodsmentioning

confidence: 99%

Section: Perspectivesmentioning

confidence: 99%

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Genome-wide SWAp-Tag yeast libraries for proteome exploration

et al. 2018

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“…The mitochondrial-localized proteins of the budding yeast Saccharomyces cerevisiae, a model of eukaryotic cells, are examples of proteins that have been extensively investigated by such approaches. Proteins in fractionated mitochondria have been systematically identified by mass spectrometry (Morgenstern et al, 2017;Reinders et al, 2006;Sickmann et al, 2003;Vögtle et al, 2017) and the subcellular localization of fluorescent protein fusion proteins has been analyzed by fluorescence microscopy for nearly all proteins (Breker et al, 2013;Chong et al, 2015;Huh et al, 2003;Koh et al, 2015;Meurer et al, 2018;Weill et al, 2018). A complementary approach to the experimental approach is the prediction of localization by bioinformatics.…”

Section: Introductionmentioning

confidence: 99%

Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm

Horiuchi

Namba

Saeki

et al. 2021

Yeast

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Eukaryotic cells are composed of organelles, and each organelle contains proteins that play a role in its function. Therefore, the localization of a protein, especially to organelles, is a clue to infer the function of that protein. In this study, we attempted to identify novel mitochondrially localized proteins in the budding yeast Saccharomyces cerevisiae using a fluorescent protein (GFPdeg) that is rapidly degraded in the cytoplasm. Of the budding yeast proteins predicted to localize to mitochondria by the prediction tool Deeploc-1.0, those with known mitochondrial localization or functional relevance were eliminated, and 95 proteins of unknown function were selected as candidates for analysis. By forced expression of GFPdeg fusion proteins with these proteins and observation of their localization, we identified 35 uncharacterized proteins potentially localized to mitochondria (UPMs) including 8 previously identified proteins that localize to mitochondria. Most of these had no N-terminal mitochondrial localization signal and were evolutionarily young "emerging genes" that exist only in S. cerevisiae. Some of these genes were found to be upregulated during the postdiauxic shift phase when mitochondria are being developed, suggesting that they are actually involved in some mitochondrial function.

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“…It is possible that there are further affected proteins which would only be revealed if a stronger overexpression was employed. Furthermore, since many proteins only tolerate either an N-or a C-terminal fusion to a tag without being functionally perturbed, a similar screen with a C-terminally GFP-tagged library may reveal even further hits (Meurer et al, 2018). Nevertheless, for TA proteins, the N-terminal fusion library that I screened is ideally suited since C-terminal GFP fusion abolishes the TA topology.…”

Section: Direct and Indirect Effects Of Loss Of Get3mentioning

confidence: 99%

The client spectrum of Get3, an evolutionarily conserved chaperone of membrane proteins

Farkas¹

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Tail-anchored proteins constitute a physiologically important class of membrane proteins. Due to their topology, they are targeted post-translationally to membranes.Although Get3 is responsible for the membrane targeting of several ER-bound TA proteins, our knowledge on its in-vivo client spectrum remains limited. The oxidationinduced chaperone activity of Get3 detected in-vitro suggests that Get3 may handle further substrates besides TA proteins. My bioinformatic analysis shows that Get3-like chaperones are more widespread than previously expected and suggests that a general chaperone activity is the ancestral function of Get3. Using budding yeast as a model system, I show that although Get3 can target many TA proteins, only a fraction of them are obligate substrates of Get3. I provide evidence that an essential TA protein, Sed5, is a super-client of Get3 that interacts with it before and also after membrane targeting. I also show that Get3 can target proteins with multiple transmembrane segments located proximal to their C-terminus as part of the GET pathway. Finally, I describe membrane proteins with amphipathic helices as potential chaperone substrates of Get3. Therefore, the current study significantly expands the range of potential Get3 clients in vivo and indicates that Get3 is an important chaperone of membrane proteins both before and after membrane targeting.

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A genome-wide resource for high-throughput genomic tagging of yeast ORFs

Cited by 4 publications

References 33 publications

Genome-wide SWAp-Tag yeast libraries for proteome exploration

Genome-wide SWAp-Tag yeast libraries for proteome exploration

Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm

The client spectrum of Get3, an evolutionarily conserved chaperone of membrane proteins

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