Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light.
Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.
Kaiserli
Eirini
E
Plant Science Group, Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, United Kingdom.
Sullivan
Stuart
S
Jones
Matthew A
MA
Feeney
Kevin A
KA
Christie
John M
JM
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D002853:Chromatography, Liquid; D002966:Clathrin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology / Q000528:radiation effects*; D047468:Immunoprecipitation; D008027:Light; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation / Q000528:radiation effects; D056949:Phototropins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
D029681:Arabidopsis Proteins; D002966:Clathrin; D004268:DNA-Binding Proteins; C527342:LOV1 protein, Arabidopsis; D056949:Phototropins