The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis

Abstract

Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy

“…( b ) Difference spectra of Np SRII reconstituted in DMPC nanodiscs (black) and solubilized with DDM (red) upon illumination with 505 nm LED light. ( c ) Difference spectra of Um Rh1 reconstituted in POPC nanodiscs (black) and solubilized with DDM [red, data taken from ( La Greca et al, 2022 )] upon illumination with 525 nm LED light. All spectra in this figure are normalized to the ethylenic modes of the retinal in the spectrum of solubilized Np SRII.…”

“…A hyperexpressing clone was generated by clone selection with increasing geneticin concentrations. The main culture was supplemented with all- trans -retinal addition and induced by methanol with subsequent purification as recently described ( La Greca et al, 2022 ).…”

“…Light-induced difference spectra of Ns XeR, Np SRII, and Um Rh1 in nanodiscs and detergent were recorded in transmission configuration using a Vertex 80v FTIR spectrometer (Bruker) as previously described ( La Greca et al, 2022 ). Spectra of Nm HR in nanodiscs and detergent were recorded in ATR configuration.…”

Proton Release Reactions in the Inward H+ Pump NsXeR

“…Bulk pH was monitored by a pH electrode (InLab Semi-Micro, Mettler Toledo) with and without light illumination (λ ≈ 500–1000 nm, Schott, ctd. 150 W halogen lamp with KG1 and OG515 filters) . The protonophore carbonylcyanid- m -chlorphenylhydrazon (CCCP; 30 μM) was added to the cell suspension when indicated (Figure S2).…”

“…Samples for IR experiments were prepared as previously described. 26 Briefly, a highly concentrated solution of nanodisc-reconstituted NsXeR in 10 mM NaCl and 5 mM Na 2 HPO 4 /NaH 2 PO 4 at pH 7.4 was prepared. Several microliters was placed on a BaF 2 window and gently dried under a stream of dry air.…”