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1.
Nat Methods ; 16(2): 205, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30602782

ABSTRACT

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.
J Mol Biol ; 431(3): 636-641, 2019 02 01.
Article in English | MEDLINE | ID: mdl-30550779

ABSTRACT

While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on function would promote accurate use of tags in experimental setups. Here we examine the effect of green fluorescent protein tagging at either the N or C terminus of budding yeast proteins on subcellular localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides a step toward broad investigation of protein fusion libraries.


Subject(s)
Fungal Proteins/metabolism , Green Fluorescent Proteins/metabolism , Protein Transport/physiology , Saccharomyces cerevisiae/metabolism , Luminescent Proteins/metabolism , Recombinant Fusion Proteins/metabolism
3.
Nat Methods ; 15(8): 617-622, 2018 08.
Article in English | MEDLINE | ID: mdl-29988094

ABSTRACT

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.


Subject(s)
Genome, Fungal , Genomic Library , Proteome/genetics , Saccharomyces cerevisiae/genetics , Genetic Complementation Test , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Protein Interaction Mapping , Proteome/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonucleoproteins, Small Nucleolar/genetics , Ribonucleoproteins, Small Nucleolar/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Tagged Sites
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