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1.
Nat Methods ; 16(2): 205, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30602782

RESUMO

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

2.
Cell ; 175(5): 1418-1429.e9, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30454649

RESUMO

We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.


Assuntos
Ferro/metabolismo , Metaloproteínas/metabolismo , Metformina/farmacologia , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Teste de Complementação Genética , Humanos , Metaloproteínas/genética , Saccharomyces cerevisiae/genética
3.
Nat Methods ; 15(8): 617-622, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988094

RESUMO

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.


Assuntos
Genoma Fúngico , Biblioteca Genômica , Proteoma/genética , Saccharomyces cerevisiae/genética , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Sequências Rotuladas
4.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803252

RESUMO

Protein-fragment complementation assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.


Assuntos
Testes Genéticos/métodos , Viabilidade Microbiana , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Seleção Genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Genes Reporter , Camundongos , Tetra-Hidrofolato Desidrogenase/genética
5.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803254

RESUMO

Here, we present a detailed protocol for studying in yeast cells the contingent interaction between a substrate and its multisubunit enzyme complex by using a death selection technique known as the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA). In yeast, the enzyme cytosine deaminase (encoded by FCY1) is involved in pyrimidine metabolism. The PCA is based on an engineered form of yeast cytosine deaminase optimized by directed evolution for maximum activity (OyCD), which acts as a reporter converting the pro-drug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a toxic compound that kills the cell. Cells that have OyCD PCA activity convert 5-FC to 5-FU and die. Using this assay, it is possible to assess how regulatory subunits of an enzyme contribute to the overall interaction between the catalytic subunit and the potential substrates. Furthermore, OyCD PCA can be used to dissect different functions of mutant forms of a protein as a mutant can disrupt interaction with one partner, while retaining interaction with others. As it is scalable to a medium- or high-throughput format, OyCD PCA can be used to study hundreds to thousands of pairwise protein-protein interactions in different deletion strains. In addition, OyCD PCA vectors (pAG413GAL1-ccdB-OyCD-F[1] and pAG415GAL1-ccdB-OyCD-F[2]) have been designed to be compatible with the proprietary Gateway technology. It is therefore easy to generate fusion genes with the OyCD reporter fragments. As an example, we will focus on the yeast cyclin-dependent protein kinase 1 (Cdk1, encoded by CDC28), its regulatory cyclin subunits, and its substrates or binding partners.


Assuntos
Citosina Desaminase/metabolismo , Teste de Complementação Genética , Viabilidade Microbiana , Complexos Multiproteicos/genética , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/genética , Seleção Genética , Citosina Desaminase/genética
6.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803260

RESUMO

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Análise Espaço-Temporal
8.
Methods Mol Biol ; 1342: 237-57, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26254928

RESUMO

Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.


Assuntos
Proteína Quinase CDC2/metabolismo , Citosina Desaminase/metabolismo , Ensaios Enzimáticos/métodos , Saccharomyces cerevisiae/enzimologia , Tubulina (Proteína)/metabolismo , Animais , Proteína Quinase CDC2/isolamento & purificação , Deleção de Genes , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9 , Spodoptera
9.
Cell Rep ; 7(4): 1333-40, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24813894

RESUMO

A single yeast cell contains a hundred million protein molecules. How these proteins are organized to orchestrate living processes is a central question in biology. To probe this organization in vivo, we measured the local concentration of proteins based on the strength of their nonspecific interactions with a neutral reporter protein. We first used a cytosolic reporter and measured local concentrations for ~2,000 proteins in S. cerevisiae, with accuracy comparable to that of mass spectrometry. Localizing the reporter to membranes specifically increased the local concentration measured for membrane proteins. Comparing the concentrations measured by both reporters revealed that encounter frequencies between proteins are primarily dictated by their abundances. However, to change these encounter frequencies and restructure the proteome, as in adaptation, we find that changes in localization have more impact than changes in abundance. These results highlight how protein abundance and localization contribute to proteome organization and reorganization.


Assuntos
Proteoma/metabolismo , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Mapeamento de Interação de Proteínas
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