Dynamic Traffic through the Recycling Compartment Couples the Metal Transporter Nramp2 (DMT1) with the Transferrin Receptor

Abstract

Nramp2 (natural resistance-associated macrophage protein 2, also called DMT1 and Slc11a2) is a proton-dependent cation transporter, which plays a central role in iron homeostasis. To study the subcellular distribution and dynamics of the transporter, we generated a construct encoding the long splice variant of Nramp2 (isoform II) tagged with the hemagglutinin epitope on a predicted extracellular loop. Cells stably transfected with this construct revealed the presence of Nramp2 in both the plasma membrane and in an endomembrane compartment. By labeling the exofacial epitope with a pH-sensitive fluorescent indicator, we were able to establish that this variant of Nramp2 resides in a vesicular compartment with an acidic lumen (pH 6.2) and that acidification was maintained by vacuolar-type ATPases. Dual labeling experiments identified this compartment as sorting and recycling endosomes. Kinetic studies by surface labeling with 125 I-labeled antibodies established that the fraction of endomembrane Nramp2 was approximately equal to that on the cell surface. The two components are in dynamic equilibrium: surface transporters are internalized continuously via a clathrin and dynamin-dependent process, whereas endosomal Nramp2 is recycled to the plasmalemma by a phosphatidylinositol 3-kinase-dependent exocytic process. Depletion of cholesterol had no discernible effect on Nramp2 internalization, suggesting that rafts or caveolae are not essential. Because the pH at the cell surface and in endosomes differs by >1 unit, the rates of transport of Nramp2 at the surface and in endomembrane compartments will differ drastically. Their subcellular colocalization and parallel trafficking suggest that Nramp2 and transferrin receptors are functionally coupled to effect pH-dependent iron uptake across the endosomal membrane.

Evolution of Differences in Transport Function in Slc11a Family Members

“…This is confirmed by studies showing that Slc11a2 and the transferrin receptor colocalize in acidic endosomes and are functionally coupled to effect pH-dependent iron uptake across the endosomal membrane (21). In contrast, Slc11a1 would be predicted to flux divalent cations from the cytoplasm into the acidic late endosomal/lysosomal compartments against the proton gradient.…”

A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles via a Two-step Mechanism

“…Alternatively, it could be because of functional coupling between the H ϩ :polyamine antiporters residing in those vesicles and the progressive decrease in pH observed along the pathway leading from MVBs (or sorting endosomes) to lysosomes (24). Such coupling has been reported to account for the tight association between the H ϩ -dependent metal symporter DMT1 and transferrin receptors in the recycling compartment (53).…”

Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responses

“…The recycling pathway is divided into: (i) the fast recycling of proteins to the plasma membrane directly from the sorting endosome (Baravalle et al 2005) and (ii) the slow recycling of proteins to the plasma membrane, which requires transit through the tubular, perinuclear endosomal recycling compartment (Sakai et al 1998). Trafficking through the endosomal recycling compartment has been extensively described for transferrin and the transferrin receptor (Touret et al 2003;Bartz et al 2005), and is also known as the constitutive recycling pathway (Ullrich et al 1996;Ren et al 1998). …”