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

• Main Authors: Shirley Renee L., Richards M. Rachel, Culbertson Michael R.
• Format: Article
• Language: English
• Published: BMC 2004-01-01
• Series: Biological Procedures Online
• Subjects: Recombination, Genetic; Gene Expression Regulation, Fungal; RNA Processing, Post-Transcriptional
• Online Access: http://www.biologicalprocedures.com/bpo/arts/1/91/m91.htm

<p>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 <it>en masse</it> to the C-terminus of variant Upf3 proteins using <it>loxP</it> sites recognized by bacterial <it>cre</it>-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.