Metal binding to the Polaris protein associated with ethylene sensing by plants

Abstract

Copper ions are essential to life, but toxic if not tightly regulated. In the model organism Arabidopsis thaliana, the ER-localised ethylene receptor, ETR1, requires Cu(I) at an intramembrane site, dependent on the Cu(I)-transporting P-type ATPase RAN1. However, the detailed biochemical mechanisms of Cu(I)-delivery, and ethylene binding, are unknown.
The protein Polaris (PLS), a negative regulator of ethylene signalling, shares some characteristics of known Cu(I)-metallochaperones, and was proposed to be involved in correct Cu(I)-metalation of ETR1. Here, metal binding to PLS has been investigated in-vitro, allowing prediction of its likely metalation state in-vivo. PLS bound Cu(I) and Zn(II) in 2:1 protein:metal stoichiometries, with β2 affinities of 3.79 x1019 and 3.76 x1012 M-2 respectively. Recently developed metalation calculators, based on metal-availability read-out from calibrated bacterial cells, were adapted to use these constants. The metal affinities of the Arabidopsis cytosolic Cu(I) chaperone Atx1, showed Cu(I) bound in a 1:1 and Zn(II) a 2:1 stoichiometry, and its metalation was modelled. This work showed, in E. coli BL21(DE3), by reading out CueR-dependent copA transcripts, Atx1 overexpression decreased Cu(I)-availability, when calibrated using E. coli JM109, with implications for heterologous expression of metalloproteins in bacteria. Availabilities, measured here, were used to correctly predict the metal preference of Atx1 in E. coli, when tested post-extraction. Using the Atx1 Cu(I)-affinity of 5.47 x10-18 M as an estimate for the intracellular buffered Cu(I)-availability in the cytosol of Arabidopsis, the metalation of PLS as a function of Atx1 Cu(I)-metalation showed it was unlikely PLS extracts Cu(I) directly from the buffer, at least not as a 2:1 complex.
This thesis speculates upon the putative roles of PLS in the biochemical activities of ETR1, and considers some of the implications and challenges associated with the potential formation of metal-dependent 2:1 ligand:metal complexes (with analogy to PLS2:Cu(I)) in biological systems, more broadly.