Evidence for mitochondrial localization of divalent metal transporter 1 (DMT1)

Abstract

In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent metal transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split-ubiquitin yeast 2-hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6-kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage-dependent anion-selective channel protein-1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the metal into the mitochondria.

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells

“…1) methodology was described in detail in previous studies (34,37). Briefly, MYTH is based on the "split ubiquitin system" (38 -41) in which ubiquitin can be split into N-terminal (Nub) and C-terminal (Cub) halves.…”

“…The technology and the bait vector pCCW-stehGLP1R-cub were described in previous studies (34,37). Briefly, the bait and prey vector were co-transformed into Saccharomyces cerevisiae host THY.AP4 strain, and colony selection was performed on yeast minimal media/synthetic defined agar plates deficient of tryptophan, leucine, histidine, and adenine negative (ϪTrp/ϪLeu/ϪHis/ϪAde).…”

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

“…9 When rat or mouse DMT1 was overexpressed in HEK293 cells under the control of a Tet-on promoter, doxycycline induction resulted in an increase of DMT1 not only in whole cell homogenates, but also in the mitochondrial fraction, as determined by immunoblotting. Moreover, DMT1 immunostaining of mitochondria isolated from these cells also increased.…”

“…1 and 2; see also ref. 9 ) relied on detecting mitochondria where their size nears the limits of fluorescence microscopy, we also tried to assess the mitochondria by flow cytometry, again extending the technical limits of the detection method. Figure 3 displays results for uninduced HEK293 cells and HEK293 cells where DMT1 has been induced by 25 nM doxycycline with panels A-E exhibiting results for rat 1A/CIRE DMT1 while panels G-K display similar results for mouse 1B/-IRE DMT1 under the same set of conditions.…”

“…6 The divalent metal transporter 1 (DMT1) is well established as a participant in iron influx into the duodenum, as the major contributor to iron efflux from the endosome during the transferrin (Tf) cycle and in handling some Tf-independent entry of iron and other metals (including copper and manganese) into many cell types. 1,2,7,8 Recently, we have reported 9 multiple lines of evidence for the presence of DMT1 in the outer mitochondrial membrane (OMM) in several cell lines and tissues from multiple origins. Experimental support came from yeast-2-hybrid, coimmunoprecipitation and immunoblotting…”