Journal ArticleDOI
A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.
Carsten Janke,Maria M. Magiera,Nicole Rathfelder,Christof Taxis,Simone Reber,Hiromi Maekawa,Alexandra C. Moreno-Borchart,Georg Doenges,Etienne Schwob,Elmar Schiebel,Michael Knop,Michael Knop +11 more
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TLDR
Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible.Abstract:
Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.read more
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Journal ArticleDOI
Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions
Chunaram Choudhary,Chanchal Kumar,Florian Gnad,Michael L. Nielsen,Michael Rehman,Tobias C. Walther,Jesper V. Olsen,Matthias Mann +7 more
TL;DR: A proteomic-scale analysis of protein acetylation suggests that it is an important biological regulatory mechanism and the regulatory scope of lysine acetylations is broad and comparable with that of other major posttranslational modifications.
Journal ArticleDOI
An auxin-based degron system for the rapid depletion of proteins in nonplant cells
TL;DR: The auxin-inducible degron (AID) system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.
Journal ArticleDOI
Progress in Metabolic Engineering of Saccharomyces cerevisiae
TL;DR: The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement and summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways.
Journal ArticleDOI
Atg9 vesicles are an important membrane source during early steps of autophagosome formation.
Hayashi Yamamoto,Soichiro Kakuta,Tomonobu M. Watanabe,Akira Kitamura,Takayuki Sekito,Chika Kondo-Kakuta,Rie Ichikawa,Masataka Kinjo,Yoshinori Ohsumi +8 more
TL;DR: Atg9-containing vesicles assemble to the preautophagosomal structure and eventually are incorporated into the autophagosome outer membrane.
Journal ArticleDOI
Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus
Keisuke Mochida,Yu Oikawa,Yayoi Kimura,Hiromi Kirisako,Hisashi Hirano,Yoshinori Ohsumi,Hitoshi Nakatogawa +6 more
TL;DR: The identification of two novel proteins, Atg39 and Atg40, as receptors specific to selective autophagy of the endoplasmic reticulum and nucleus in the yeast Saccharomyces cerevisiae provides fundamental insight into the pathophysiological roles and mechanisms of ‘ER-phagy’ and ‘nucleophagy” in other organisms.
References
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TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Journal ArticleDOI
Functional organization of the yeast proteome by systematic analysis of protein complexes
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Journal ArticleDOI
Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry
Yuen Ho,Albrecht Gruhler,Adrian Heilbut,Gary D. Bader,Gary D. Bader,Lynda Moore,Sally-Lin Adams,Anna Millar,Paul J. Taylor,Keiryn L. Bennett,Kelly Boutilier,Lingyun Yang,Cheryl Wolting,Ian Donaldson,Søren Schandorff,Juanita Shewnarane,Mai Vo,Joanne Taggart,Marilyn Goudreault,Brenda Muskat,Cris Alfarano,Danielle Dewar,Zhen Lin,Katerina Michalickova,Katerina Michalickova,Andrew Willems,Andrew Willems,Holly Sassi,Peter A Nielsen,Karina Juhl Rasmussen,Jens R. Andersen,Lene E. Johansen,Lykke Haastrup Hansen,Hans Jespersen,Alexandre V. Podtelejnikov,Eva Nielsen,Janne S. Crawford,Vibeke Poulsen,Birgitte D Sørensen,Jesper Matthiesen,Ronald C. Hendrickson,Frank Gleeson,Tony Pawson,Tony Pawson,Michael Moran,Daniel Durocher,Daniel Durocher,Matthias Mann,Christopher W. V. Hogue,Christopher W. V. Hogue,Daniel Figeys,Mike Tyers,Mike Tyers +52 more
TL;DR: Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies.
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