MondoA-Mlx heterodimers are candidate sensors of cellular energy status: mitochondrial localization and direct regulation of glycolysis

Abstract

Transcription factors can be sequestered at specific organelles and translocate to the nucleus in response to changes in organellar homeostasis. MondoA is a basic helix-loop-helix leucine zipper transcriptional activator similar to Myc in function. However, unlike Myc, MondoA and its binding partner Mlx localize to the cytoplasm, suggesting tight regulation of their nuclear function. We show here that endogenous MondoA and Mlx associate with mitochondria in primary skeletal muscle cells and erythroblast K562 cells. Interaction between MondoA and the mitochondria is salt and protease sensitive, demonstrating that it associates with the outer mitochondrial membrane by binding a protein partner. Further, endogenous MondoA shuttles between the mitochondria and the nucleus, suggesting that it communicates between these two organelles. When nuclear, MondoA activates transcription of a broad spectrum of metabolic genes, including those for the glycolytic enzymes lactate dehydrogenase A, hexokinase II, and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3. Regulation of these three targets is mediated by direct interaction with CACGTG sites in their promoters. Consistent with its regulation of glycolytic targets, MondoA is both necessary and sufficient for glycolysis. We propose that MondoA communicates information about the intracellular energy state between the mitochondria and the nucleus, resulting in transcriptional activation of glycolytic target genes.

FIG. 1.

MondoA and Mlx colocalize with the mitochondria. (A) Primary human SkMC were labeled with anti-MondoA antibody and MitoTracker, and the two images were merged. (B) Primary human SkMC were labeled with anti-Mlx and anti-cytochrome c antibodies, and the two images were merged. The images in panels A and B were obtained by confocal microscopy.

FIG. 2.

MondoA is enriched in the mitochondrial fraction from SkMC and K562 cells. SkMC (A) or K562 cells (B) were separated into nuclear (Nuc), cytoplasmic (Cyto), and heavy membrane (HM) fractions. Levels of MondoA, Mlx, the nuclear markers mSin3A and mSDS3, and the mitochondrial marker cytochrome c were determined by Western blotting. Mitochondria were purified by sucrose density gradient centrifugation (C), and the levels of MondoA, the mitochondrial marker porin, the Golgi marker Golgin-97, and the endosomal/lysosomal marker mannose-6-phosphate receptor (M-6-P) were determined by Western blotting. WCE, whole cell extract; Mito; mitochondrial fraction.

FIG. 3.

MondoA associates with an OMM protein. (A) Mitochondria were prepared from K562 cells and were mock treated or incubated with proteinase K in the presence or absence of Triton X-100 as indicated. Levels of MondoA, the outer membrane marker porin, and the intermembrane space marker cytochrome c were determined by Western blotting. WCE, whole cell extract; Mito; mitochondrial fraction. (B) Isolated mitochondria from K562 cells were incubated with a cytoplasmic lysate containing MondoA-TAP in the presence of increasing KCl concentrations. After binding and washing, the amounts of MondoA, MondoA-TAP, and aconitase that remained associated with mitochondria were determined by Western blotting. (C) Isolated mitochondria were left untreated (NT), treated with PMSF, treated with proteinase K followed by PMSF, or treated with PMSF followed by proteinase K. Treated mitochondria were incubated with a cytoplasmic lysate containing MondoA-TAP. Following binding and washing of the mitochondrial pellets, the amount of MondoA, MondoA-TAP (bound), or porin (Mito Input) that remained associated with mitochondria following each treatment was determined by Western blotting.

FIG. 4.

The association of MondoA and the mitochondria is dynamic. (A) SkMC were either treated with 10 ng/ml of the nuclear export inhibitor LMB for 4 h or left untreated. Cells were labeled with anti-MondoA antibody, and images were obtained by confocal microscopy. (B) The subcellular localization of endogenous MondoA in SkMC was quantified in a double-blind experiment. Nuclear, predominantly nuclear labeling; mitochondria + nuclear, equivalent mitochondria and nuclear labeling; mitochondria, predominantly mitochondria labeling; Un, untreated.

FIG. 5.

MondoA regulates the transcription of glycolytic enzymes. (A) Expression of ΔN237NLSMondoA and ΔN237NLSMondoA(H724P) in C2C12 cells was determined by Western blotting. The HKII, PFKFB3, and LDH-A levels (B) and glycolysis rates (C) in C2C12 cells expressing ΔN237NLSMondoA or ΔN237NLSMondoA(H724P) or infected with pBabePuro alone were determined.

FIG. 6.

MondoA can regulate glycolytic genes directly. (A) The activities of the wild-type (WT) and double CACGTG E-box mutant (mE1 + mE2) LDH-A luciferase reporters were measured in C2C12 cells expressing the indicated combinations of ΔN237MondoA (MondoA) and NLS-Mlx (Mlx) or the corresponding empty expression vectors. (B) Schematic diagrams of the LDH-A, HKII, and PFKFB3 promoters. Exons (E) and transcriptional start sites (+1) and locations of CACGTG E-box elements are indicated. Lines above the promoters indicate the regions amplified by PCR. (C) Real-time PCR assay measuring enrichment of ChIPs from C2C12 cells expressing ΔN237NLSMondoA performed with MondoA, c-Myc, or Gal4p antibodies. Immunoprecipitated DNA was analyzed by real-time PCR with primers specific to the E-box-containing regions of the mouse LDH-A, HKII, and PFKFB3 promoters. Measurements are expressed as fold enrichment over the amount of an upstream region of the PFKFB3 promoter that does not contain a CACGTG site. Each value is the average (±the standard error) from two independent biological replicates.

FIG. 7.

Endogenous MondoA regulates glycolysis. (A) Real-time PCR measured enrichment of ChIPs from BJ fibroblasts performed with MondoA, c-Myc, or Gal4p antibodies. Immunoprecipitated DNA was analyzed by real-time PCR with primers specific to the E-box-containing regions of the human LDH-A, HKII, and PFKFB3 promoters. Measurements are expressed as fold enrichment over the amount of an upstream region of the PFKFB3 promoter that does not contain a CACGTG site. Each value is the average (±the standard error) from at least three independent biological replicates. Expression levels of MondoA and tubulin (B) and glycolysis rates (C) in K562 cells expressing the indicated shRNA constructs are also shown. NS, a scrambled shRNA that does not recognize any sequence in the human genome; M1 and M2, shRNA sequences specific for human MondoA.