Question:

In renal proximal tubules transferrin-bound iron is thought to be internalized by receptor-mediated endocytosis and trafficked to acidic lysosomes where efflux of free iron occurs via divalent metal transporter 1 (DMT1) for storage, transport and/or metabolic use, e.g. in mitochondria. To prevent toxicity due to Fenton chemistry, iron may be shuttled to mitochondria by cytosolic chaperones or delivered directly by a “kiss-and-run” docking mechanism. We therefore aimed to identify putative renal DMT1 interaction partners.

Methods:

A human kidney cDNA prey library was probed with human DMT1 (hDMT1) as bait by split-ubiquitin yeast two-hybrid screening. Subcellular protein localization was assayed by confocal microscopy, co-immunoprecipitation, immunoblotting of percoll gradient-purified rat kidney cortical mitochondria, and immunogold labeling of rat renal tissue.

Results:

Two mitochondrial proteins were identified as interaction partners, namely cytochrome C oxidase subunit II (COXII) and translocase of the outer mitochondrial membrane (OMM) subunit 6 (Tom6). In HEK293 cells, COXII co-immunoprecipitated with endogenous hDMT1, and with hDMT1-FLAG from hDMT1-overexpressing but not mock-transfected cells. Overexpressed hDMT1-FLAG partially co-localized with endogenous COXII as well as Tom6. In purified mitochondria, enrichment of DMT1 correlated with the mitochondrial proteins VDAC1 and ANT, but not with the lysosomal marker Lamp1. Mitoplast preparations indicated that DMT1 is localized in the OMM. Accordingly, immunogold labeling indicated co-localization of DMT1 and VDAC1 in OMM.

Conclusions:

Thus, in addition to lysosomes, DMT1 appears to be localized in the OMM, where it may be involved in transport of iron and possibly other essential metals for mitochondrial function.

Funding:

DFG TH345/11-1