Comprehensive approaches to detect protein-protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p-Mms1p and Sae2p-Mre11p. The Rtt101p-Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies.
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http://dx.doi.org/10.1101/gr.6667007 | DOI Listing |
Microorganisms
December 2024
Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Carrer de les Sitges, s/n, 08193 Bellaterra, Catalonia, Spain.
The introduction of heterologous pathways into microbial cell compartments offers several potential advantages, including increasing enzyme concentrations and reducing competition with native pathways, making this approach attractive for producing complex metabolites like fatty acids and fatty alcohols. However, measuring subcellular concentrations of these metabolites remains technically challenging. Here, we explored 3-hydroxypropionic acid (3-HP), readily quantifiable and sharing the same precursors-acetyl-CoA, NADPH, and ATP-with the above-mentioned products, as a reporter metabolite for peroxisomal engineering in the yeast .
View Article and Find Full Text PDFISME Commun
January 2024
Otto-von-Guericke University Magdeburg, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Saxony-Anhalt, Germany.
A comprehensive understanding of microbial community dynamics is fundamental to the advancement of environmental microbiology, human health, and biotechnology. Metaproteomics, defined as the analysis of all proteins present within a microbial community, provides insights into these complex systems. Microbial adaptation and activity depend to an important extent on newly synthesized proteins (nP), however, the distinction between nP and bulk proteins is challenging.
View Article and Find Full Text PDFJ Cell Biol
February 2025
Institute of Molecular Biology, Mainz, Germany.
Functional genomics with libraries of knockout alleles is limited to non-essential genes and convoluted by the potential accumulation of suppressor mutations in knockout backgrounds, which can lead to erroneous functional annotations. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for inducible degradation of AID-tagged proteins in the budding yeast Saccharomyces cerevisiae. First, we determined that N-terminal tagging is at least twice as likely to inadvertently impair protein function across the proteome.
View Article and Find Full Text PDFJ Cell Biol
February 2025
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Genome-wide collections of yeast strains, known as libraries, revolutionized the way systematic studies are carried out. Specifically, libraries that involve a cellular perturbation, such as the deletion collection, have facilitated key biological discoveries. However, short-term rewiring and long-term accumulation of suppressor mutations often obscure the functional consequences of such perturbations.
View Article and Find Full Text PDFG3 (Bethesda)
December 2024
Laboratory of the Physics of Biological Systems, Institute of Physics, École Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
Scar-less genome editing in budding yeast with elimination of the selection marker has many advantages. Some markers such as URA3 and TRP1 can be recycled through counterselection. This permits seamless genome modification with pop-in/pop-out (PIPO), in which a DNA construct first integrates in the genome and, subsequently, homologous regions recombine and excise undesired sequences.
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