Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells.
Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.
Ferrando
A
A
Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany.
Koncz-Kálmán
Z
Z
Farràs
R
R
Tiburcio
A
A
Schell
J
J
Koncz
C
C
D017360:Arabidopsis / Q000166:cytology / Q000235:genetics*; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression; D016259:Genes, myc / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006388:Hemagglutinins / Q000235:genetics; D007438:Introns / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
D000939:Epitopes; D006388:Hemagglutinins; D008164:Luminescent Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D005966:Glucuronidase