Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1.
The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1-349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (K(d), 117 nM) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (K(d), 1.38 μM). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
Chun-Hai
CH
Dong
Mihue
M
Jang
Benjamin
B
Scharein
Anuschka
A
Malach
Maximo
M
Rivarola
Jeff
J
Liesch
Georg
G
Groth
Inhwan
I
Hwang
Caren
C
Chang
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005030:Ethylenes / Q000378:metabolism*; D005816:Genetic Complementation Test; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; C494590:ETR1 protein, Arabidopsis; D005030:Ethylenes; D008565:Membrane Proteins; C525374:RTE1 protein, Arabidopsis; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C036216:ethylene