Abstract
The plant hormone ethylene is a key regulator of plant growth, development and stress adaption. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs) localized at the ER-Golgi network. The biological function of these receptors relies on a protein-bound copper cofactor. Nonetheless, molecular processes and structures controlling assembly and integration of the metal into the functional plant hormone receptor are still unknown. Here, we have explored the molecular pathways of copper transfer from the plant cytosol to the ethylene receptor family by analyzing protein–protein interactions of receptors with soluble and membrane-bound plant copper carriers. Our results suggest that receptors primarily acquire their metal cofactor from copper transporter RESPONSIVE-TO-ANTAGONIST-1 (RAN1) which has been loaded with the transition metal beforehand by soluble copper carriers of the ATX1-family. In addition, we found evidence for a direct interaction of ETRs with soluble chaperones ANTIOXIDANT-1 (ATX1) and COPPER TRANSPORT PROTEIN (CCH) raising the possibility of a direct copper exchange between soluble chaperones and receptors.
Highlights
Dilution series of ATX1, CCH and CCHΔ from 30 μM to 0.9 nM were prepared in buffer SEC (50 mM HEPES, 300 mM NaCl, pH 7.5)
NterRAN1 was diluted to a concentration of 40 nM in buffer SEC (50 mM HEPES, 300 mM NaCl, pH 7.5) containing 0.05% (w/v) Tween[20]
A serial dilution of ETR11–157 from 7.5 μM to 0.2 nM was prepared in buffer SEC (50 mM HEPES, 300 mM NaCl, pH 7.5) containing 0.015% (w/v) FosCholine-16 and 0.05% (w/v) Tween[20]
Summary
500 μl were loaded on a Superdex200 10/300 GL size exclusion column, equilibrated with buffer SEC (50 mM HEPES, 300 mM NaCl, pH 7–8.2) or buffer SEC + 0.1% (w/v) DDM. For interaction studies by microscale thermophoresis, recombinant proteins were labeled with AlexaFluor488-NHS (Life Technologie) prior the final purification step by size exclusion. 500 μl of labeled protein solution was loaded on a Superdex200 10/300 GL size exclusion column to separate aggregates, TEV protease and free dye from the protein of interest in a single step.
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