MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity

Abstract

Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252-E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X(2)-(S/T)-X(3)-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates.

Fig. 1.

A mitoNEET protein family. Alignment of the amino acid sequences of human mitoNEET, Miner1, and Miner2. Amino acids conserved in humans are shown in gray. Amino acids invariant across multiple species are indicated in yellow. The CDGSH domains are noted by red bars. The transmembrane domain of mitoNEET is marked by a blue box.

Fig. 2.

Localization of MitoNEET family proteins. (A–C) Immunofluorescent staining of V5-tagged mitoNEET (A), Miner1 (B), and Miner2 (C) transiently transfected for 24 h in COS-7 cells. MitoNEET family proteins appear green. Colocalization of mitoNEET and Miner2 (Left) with MitoTracker Red (MR) (Center) appears yellow in the merged images (Right). DNA is stained blue with DAPI. (D) Immunofluorescent imaging in COS-7 cells of endogenous mitoNEET, MitoTracker Red, and merged images.

Fig. 3.

MitoNEET is directed to the OMM by an amino-terminal signal sequence. (A–D) Immunofluorescent imaging of transiently expressed mitoNEET proteins in COS-7 cells: V5-tagged full-length mitoNEET (A), V5-tagged mitoNEET lacking amino acids 1–32 (B), EGFP (C), and EGFP with amino acids 1–32 of mitoNEET fused to the amino terminus (D). (E) Immunogold electron microscopic staining of mitochondria in rat kidney tissue sections with either preimmune serum (PI) or anti-mitoNEET antibody. (F) Rat liver mitochondrial subfractions were separated by SDS/PAGE and analyzed by immunobloting by using antibodies against known mitochondrial marker proteins. Abbreviations: SMP, submitochondrial particles; IMS, intermembrane space; VDAC, voltage-dependent anion channel; SMAC, second mitochondrial activator of caspases; PTPMTI, protein tyrosine phosphatase of the mitochondria-1.

Fig. 4.

MitoNEET is an integral membrane protein oriented toward the cytoplasmic face of the OMM. (A) Immunoblot analysis of swollen rat liver mitochondria treated to remove peripheral membrane proteins. Mitochondria were washed with either 200 mM KCl (high salt) or 0.1 M Na₂CO₃, pH 11.5 (high pH), and the proteins remaining in the pellets were separated by SDS/PAGE and immunoblotted. (B) SDS/PAGE and immunoblot analysis of trypsin-treated rat heart mitochondria (osmotically shocked). (C) SDS/PAGE and immunoblot analysis of trypsin-treated rat heart mitochondria (low pH washed). BCL2, B cell lymphoma-2; SMAC, second mitochondrial activator of caspases; VDAC, voltage-dependent anion channel protein.

Fig. 5.

MitoNEET protein levels affect maximal mitochondrial oxidative capacity. (A) Respiration rates of cardiac mitochondria from mitoNEET (+/+) (filled bars) and (−/−) (open bars) mice. Error bars represent the standard deviation from n = 7 (+/+) and n = 6 (−/−) from three separate preparations (P < 0.01). (B) SDS/PAGE and immunoblot analysis of purified cardiac mitochondria from mitoNEET (+/+) and (−/−) mice. NDUFB6 is a subunit of complex I.