Open Access

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

  • Renee L. Shirley1,
  • M. Rachel Richards1 and
  • Michael R. Culbertson1Email author
Biological Procedures Online6:610209

DOI: 10.1251/bpo91

Received: 9 July 2004

Accepted: 24 September 2004

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.

Indexing terms

Recombination, Genetic Gene Expression Regulation, Fungal RNA Processing, Post-Transcriptional

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