Cloning by pathway activation in yeast: identification of an Arabidopsis thaliana F-box protein that can turn on glucose repression.
We describe a method for identifying signal transducing proteins from other organisms by their ability to turn on a signalling pathway when they are expressed at high level in yeast. The method was tested on a cDNA library from Arabidopsis thaliana, which was screened for clones that can activate glucose repression in the absence of glucose. Six clones were characterized. One of them codes for AtGRH1, a new F-box protein that shows similarity to GRR1, a yeast protein involved in glucose repression. The ability of AtGRHI to activate glucose repression is dependent on the MIG1 repressor. Two-hybrid experiments revealed that AtGRH1 can interact with AtSKP1a and AtSKP1b, two recently identified SKP1 homologues in Arabidopsis. Other clones identified in the screen encode the transcription factor AtEBP, the 14-3-3 protein AtGF14 and two new proteins: AtMYR1 and AtPOZ1. None of these proteins turn on glucose repression. Instead, they illustrate various other ways by which foreign proteins can interfere with expression of a yeast gene. We conclude that our method worked as expected in at least one case, and that it could be applied to other signalling pathways that are conserved between yeast and higher eukaryotes.
Thelander
Mattias
M
Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala.
Fredriksson
Dan
D
Schouten
Jan
J
Hoge
J Harry C
JH
Ronne
Hans
H
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005686:Galactokinase / Q000235:genetics; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D044786:S-Phase Kinase-Associated Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics*
D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D044786:S-Phase Kinase-Associated Proteins; D000242:Cyclic AMP; D005686:Galactokinase; D005947:Glucose