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Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins

Biological Procedures Online volume 6, pages 209–219 (2004)Cite this article

Abstract

A method was developed to assess the functional significance of a sequence motif in yeast Upf3p, a protein required for nonsense-mediated mRNA decay (NMD). The motif lies at the edge of the Upf3p-Upf2p interaction domain, but at the same time resembles the canonical leucine-rich nuclear export sequence (NES) found in proteins that bind Crm1p exportin. To test the function of the putative NES, site-directed mutations that cause substitutions of conserved NES-A residues were first selected to identify hypermorphic alleles. Next, a portable Crm1p-binding NES from HIV-1 Rev protein that functions in yeast was fused en masse to the C-terminus of variant Upf3 proteins using loxP sites recognized by bacterial cre-recombinase. Finally, variant Upf3-Rev proteins that were functional in NMD were selected and examined for the types of amino acid substitutions present in NES-A. The mutational analysis revealed that amino acid substitutions in the Upf3 NES impair both nuclear export and the Upf2p-Upf3p interaction, both of which are required for Upf3p to function in NMD. The method described in this report could be modified for the genetic analysis of a variety of portable protein domains.

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Authors and Affiliations

  1. Laboratory of Molecular Biology, R.M. Bock Labs, University of Wisconsin, 1525 Linden Drive, 53706, Madison, WI

    Renee L. Shirley, M. Rachel Richards & Michael R. Culbertson

Authors
  1. Renee L. Shirley
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  2. M. Rachel Richards
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  3. Michael R. Culbertson
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Correspondence to Michael R. Culbertson.

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Published: October 1, 2004.

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Shirley, R.L., Richards, M.R. & Culbertson, M.R. Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins. Biol. Proced. Online 6, 209–219 (2004). https://doi.org/10.1251/bpo91

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  • DOI: https://doi.org/10.1251/bpo91

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Biological Procedures Online

ISSN: 1480-9222

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