Prp8p dissection reveals domain structure and protein interaction sites.
We describe a novel approach to characterize the functional domains of a protein in vivo. This involves the use of a custom-built Tn5-based transposon that causes the expression of a target gene as two contiguous polypeptides. When used as a genetic screen to dissect the budding yeast PRP8 gene, this showed that Prp8 protein could be dissected into three distinct pairs of functional polypeptides. Thus, four functional domains can be defined in the 2413-residue Prp8 protein, with boundaries in the regions of amino acids 394-443, 770, and 2170-2179. The central region of the protein was resistant to dissection by this approach, suggesting that it represents one large functional unit. The dissected constructs allowed investigation of factors that associate strongly with the N- or the C-terminal Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437-770, whereas fragmenting Prp8p at residue 2173 destabilizes its association with Aar2p.
Boon
Kum-Loong
KL
Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
Norman
Christine M
CM
Grainger
Richard J
RJ
Newman
Andrew J
AJ
Beggs
Jean D
JD
D001483:Base Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C440573:Aar2p protein, S cerevisiae; D004251:DNA Transposable Elements; D009687:Nuclear Proteins; C077018:PRP8 protein, S cerevisiae; D011994:Recombinant Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; C472240:SNU114 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins