Abstract
The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.
Highlights
The ATP-binding cassette (ABC)5 protein superfamily is one of the largest protein families known, and many but not all are membrane proteins (“ABC transporters”) competent in the transport of a broad range of materials across membranes
Total RNA was isolated from 15-day-old Arabidopsis seedlings that had been grown in standard liquid MS medium, in the case of AtATM1 and AtATM3, or in liquid MS medium containing 50 M CdCl2, in the case of AtATM2, using TriZol Reagent (Invitrogen) according to the manufacturer’s recommendations. cDNA was synthesized from the RNA extracts using the SuperScript Preamplification System (Invitrogen)
Cloning and Sequence Analysis of AtATM1, AtATM2, and AtATM3—There are three ORFs, designated AtATM1, AtATM2, and AtATM3, in the genome of Arabidopsis capable of encoding proteins bearing a close similarity to S. cerevisiae ATM1 (ScATM1) [2]
Summary
The ATP-binding cassette (ABC) protein superfamily is one of the largest protein families known, and many but not all are membrane proteins (“ABC transporters”) competent in the transport of a broad range of materials across membranes. ABC proteins are designated as such, because each possesses one or two ATP-binding cassettes or nucleotide binding folds (NBFs) sharing 30 – 40% identity between family members and one or more transmembrane domains (TMDs) [1]. One of the few exceptions is the AtATM (A. thaliana“ABC transporter of the mitochondrion” homolog) subfamily of forward orientation half-molecule ABC transporters. It contains only three ORFs: two that are immediately adjacent to each other on chromosome IV (AtATM1 and AtATM2) and one other on chromosome V (AtATM3) [2, 5].6. The abbreviations used are: ABC, ATP-binding cassette; GFP, green fluorescent protein; NBF, nucleotide-binding fold; ORF, open reading frame; TMD, transmembrane domain; TP, targeting peptide; RT, reverse transcription. AtATM Subfamily of ABC Transporters (Eugene 3.00131305 and Genewise1-v1.C_LG_XIX2542), all three of which most closely resemble AtATM3, have been assigned to this subfamily in the genomes from other plant sources [3].7
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