Mitoferrin-2 (MFRN2) Regulates the Electrogenic Mitochondrial Calcium Uniporter and Interacts Physically with MCU

Abstract

Background: The mitochondrial calcium uniporter catalyzes electrogenic mitochondrial uptake of both Ca2+ and Fe2+ (JBC 1975;250:6433). More recently, Mfrn1 and 2 were identified to mediate mitochondrial iron uptake in hemopoietic and non-hemopoietic tissue, respectively (Mol Cell Biol 2009;29:1007), whereas the CCDC109A gene product (MCU) was discovered to be the core protein of the uniporter complex (Nature 2011;476:336&341). Here, our AIM was to determine the role of Mfrn2 in uniporter function. Methods: Respiration-driven Ru360-sensitive mitochondrial Fe2+ and Ca2+ uptake was measured in rat liver mitochondria (RLM) and permeabilized UMSCC1 and UMSCC22 squamous carcinoma cells. siRNA knockdown of Mfrn2 was also performed in UMSCC22A cells. Pull down assays were performed in HeLa cells expressing MCU-V5 and Mfrn2-GFP. Results: In RLM, Ca2+ and Fe2+ (250 μM) each stimulated respiration to a nearly identical degree, an effect completely blocked by Ru360. In UMSCC22A cells, mRNA and protein expression of Mfrn2 was 2-3-times that observed in UMSCC1 cells. High Mfrn2-expressing UMSCC22 cells also had 3-fold greater rates of mitochondrial Ca2+ and Fe2+ uptake. After Mfrn2 knockdown (55% decrease), rates of mitochondrial uptake of both Ca2+ and Fe2+ decreased by ∼75%. All uptakes were blocked by Ru360. In HeLa cells co-transfected with MCU-V5 and Mfrn2-GFP, anti-GFP beads pulled down MCU-V5, whereas anti-V5 beads pulled down Mfrn2-GFP. COX-IV was not pulled down by beads, indicating that the interaction between MCU and Mfrn2 was specific. Conclusions: 1) Mfrn2 positively modulates Ru360-sensitive respiration-driven mitochondrial uptake of both Ca2+ and Fe2+. 2) Mfrn2 physically interacts with MCU and appears to be a component/regulator of the MCU complex. 3) The mitochondrial calcium uniporter should more appropriately be called the mitochondrial Ca2+,Fe2+ uniporter.