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Mol Cell Proteomics. 2013 Nov;12(11):3370-8. doi: 10.1074/mcp.O113.031708. Epub 2013 Jul 29.

High-throughput analysis of in vivo protein stability.

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1
Department of Genome Sciences, University of Washington, Box 355065, Seattle, Washington 98195;

Abstract

Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with high-throughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein. The variants are fused to a metabolic enzyme, which here is the yeast Leu2 protein. Plasmids encoding these Leu2 fusion proteins are transformed into yeast, with the resultant fusion proteins accumulating to different levels based on their stability and leading to different doubling times when the yeast are grown in the absence of leucine. Sequencing of an input population of variants of a protein and the population of variants after leucine selection allows the stability of tens of thousands of variants to be scored in parallel. By applying the Stable-seq method to variants of the protein degradation signal Deg1 from the yeast Matα2 protein, we generated a high-resolution map that reveals the effect of ∼30,000 mutations on protein stability. We identified mutations that likely affect stability by changing the activity of the degron, by leading to translation from new start codons, or by affecting N-terminal processing. Stable-seq should be applicable to other organisms via the use of suitable reporter proteins, as well as to the analysis of complex mixtures of fusion proteins.

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PMID:
23897579
PMCID:
PMC3820947
DOI:
10.1074/mcp.O113.031708
[Indexed for MEDLINE]
Free PMC Article

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