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. 2022 Dec 31;17(1):2005881.
doi: 10.1080/15592324.2021.2005881. Epub 2021 Nov 23.

HRM and CRAC in MxIRT1 act as iron sensors to determine MxIRT1 vesicle-PM fusion and metal transport

Song Tan  1   2 , Xi Zhang  2 , Qi Zhang  2 , Yu-Meng Li  2 , Peng Zhang  2 , Li-Ping Yin  2
Affiliations

HRM and CRAC in MxIRT1 act as iron sensors to determine MxIRT1 vesicle-PM fusion and metal transport

Song Tan et al. Plant Signal Behav. .

Abstract

The IRON-REGULATED TRANSPORTER1 (IRT1) is critical for iron uptake in roots, and its exocytosis to the plasma membrane (PM) is regulated by detergent-resistant membranes. However, studies on IRT1 exocytosis and function in response to iron status are limited. Presently, we found that the histidine-rich motif (HRM) of MxIRT1 could bind to iron directly and HRM determined the delivery of MxIRT1 to the PM, after which the cholesterol recognition amino acid consensus (CRAC) motif-regulated MxIRT1 mediated metal transport. IMAC assay revealed that H192 was the vital site for HRM binding to Fe2+, and metal-binding activity was stopped after the deletion of HRM (MxIRT1∆HM) or in H192 site-directed mutants (H192A). MxIRT1∆HM or H192A in transgenic yeast and Arabidopsis failed to localize in the PM and displayed impaired iron absorption. In the PM, Y266 in CRAC was required for metal transport; Y266A transgenic Arabidopsis displayed the same root length, Cd2+ flux, and Fe concentration as Arabidopsis mutant irt1 under iron-deficient conditions. Therefore, H192 in HRM may be an iron sensor to regulate delivery of MxIRT1 vesicles to the PM after binding with iron; Y266 in CRAC acts as an iron sensor for active metal transport under iron-deficient conditions.

Keywords: CRAC motif; Histidine rich motif; MxIRT1; detergent-resistant membrane; iron; iron sensor.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1.
Figure 1.
Sequence alignment of His rich motif (HRM) among the loop in IRT1s and schematic representation of MxIRT1 mutants.
Figure 2.
Figure 2.
Phenotypic and functional comparison of Vector (GFP), MxIRT1ΔHM (MxIRT1ΔHM-GFP), MxIRT1 (MxIRT1-GFP) transgenic and WT (wild type) Arabidopsis thaliana.
Figure 3.
Figure 3.
Functional analysis and PM localization of MxIRT1 HRM point mutants in yeast.
Figure 4.
Figure 4.
Functional analysis and PM localization of MxIRT1 HRM point mutants in plant.
Figure 5.
Figure 5.
Iron-binding analysis of MxIRT1 HRM point mutants using immobilized metal ion affinity chromatography (IMAC).
Figure 6.
Figure 6.
Phenotypic and functional analysis of Vector (GFP), MxIRT1 CRAC motif site-directed mutagenesis (L262A-GFP, Y266A-GFP, K270A-GFP) transgenic and WT Arabidopsis thaliana under iron sufficient condition.
Figure 7.
Figure 7.
Phenotypic and functional analysis of Vector (GFP), MxIRT1 CRAC motif site-directed mutagenesis (L262A-GFP, Y266A-GFP, K270A-GFP) transgenic and WT Arabidopsis thaliana under iron deficient conditions.
See this image and copyright information in PMC

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