Abstract
Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1–29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake.
Highlights
Iron is an essential nutrient for plants and other organisms
The findings presented here clearly demonstrate that Malus xiaojinensis iron-regulated transporter 1 (Mx iron-regulated transporter 1 (IRT1)), an iron transport protein essential for plant growth, viability, and productivity, is a multipass plasma membrane (PM) protein that must be sorted to the PM via an signal peptide (SP)-dependent ER trafficking process
Mx DsIRT1 protein could not be translocated into the ER for trafficking to the PM
Summary
Iron is an essential nutrient for plants and other organisms. Previous studies have used reverse genetics to demonstrate that iron-regulated transporter 1 (IRT1) is a major multi-pass plasma membrane (PM) importer responsible for iron uptake from soil under conditions of iron deficiency [2,3,4]. The IRT1 protein family is widespread, occurring in a variety of dicots and monocots including but not limited to Arabidopsis [2], maize [5], rice [6], and Malus xiaojinensis [7]. A series of studies with Arabidopsis have provided significant new insights on the sorting, trafficking and localization of IRT1 within plant cells. Additional findings have demonstrated that members of the sorting nexin (SNX1) family of proteins are necessary for the correct trafficking of At (Arabidopsis thaliana) IRT1, and for modulating overall iron uptake activity [9].
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