Mitochondrial DNA content Mitochondrial biogenesis was assessed by determining the mitochondrial DNA copy number. Total DNA was isolated using a modification of the spin-column protocol from the DNeasy Blood and Tissue Kit (Qiagen), then the quantity of the mitochondrial encoded gene cytochrome c oxidase subunit 1 (CO1) and the quantity of the stable nuclear-encoded gene 18S ribosomal RNA (rRNA) were measured via PCR. Mitochondrial DNA copy number was expressed as the ratio of CO1 to 18SrRNA expression. Hexokinase activity The QuikChange II Multi-site Directed Mutagenesis Kit (Stratagene) was used to make point mutations in HKIII substrate-binding residues. The full-length HKIII protein was used as the template. HKIII constructs were PCR-amplified from human genomic DNA and subcloned into pET28a (Novagen). Proteins were expressed in E. coli (BL21 DE3) and purified with Ni-NTA affinity chromatography and Superdex 75 (26/60) size- exclusion chromatography (pre-equilibrated with 10 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 5 mM DTT). Proteins were concentrated using an Amicon Ultra centrifugal filter with purity .95% based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. HK activity was assessed as described previously [17] by coupling G6P production with NADP reduction in the presence of G6P dehydrogenase (G6PDH). Statistical analysis Results are presented as mean6SEM. The Student’s t-test was used to analyze data with unequal variance between each group. A p-value of less than 0.05 was considered significant. Results Hypoxia induces HKIII expression through HIF dependent signaling Analysis of the HKIII promoter sequence identified several putative regulatory elements responsive to cAMP, AMPK, or HIF signal transduction, which have been shown to regulate HKII expression [30,31,32]. Treatment of Novikoff N1S1 rat hepatoma cells, which have measurable baseline levels of HKIII expression, with the cAMP inducer forskolin or the AMPK activator AICAR did not significantly change HKIII mRNA expression levels (Figure S1A and B). However, HKIII expression significantly increased at the mRNA and protein levels in response to hypoxia (Figure 1A). Hypoxic regulation of HKIII was also demonstrated at the mRNA level in other cell types (Figure 1D). We then studied whether the regulation of HKIII by hypoxia is through a HIF-dependent pathway. Treatment of N1S1 cells under normoxic conditions with the prolyl hydroxylase inhibitor and HIF stabilizer Dimethyloxallyl glycine (DMOG) resulted in a significant increase in HKIII mRNA and protein levels (Figure 1B). Overexpression of constitutively active (ca) HIF-1a and 2a constructs, also resulted in similar increases in HKIII expression at the mRNA level compared to the vector control (Figure 1C). These constructs contain double P to A point mutations that prevent prolyl hydroxylation and subsequent degradation under normoxic conditions [29]. Overall, the results indicate that regulation of HKIII expression is partially mediated through HIF signaling, which does not appear to be isoform specific. HKII expression served as a positive control for these experiments, and the data are presented in Figure S2A and B). HK3 Regulation and Protection N-terminal substrate binding regulates HKIII activity Glucose binding to the N-terminal domain (N-half) of HKII influences the regulation of the C-half by G6P [17], demonstrating HK regulation at the post-translational level. We studied whether an intramolecular interaction between the two domains also exists in HKIII. The HKIII sequence was aligned with those of HKI and HKII to predict which residues in its N- and C- terminal halves bind glucose, ATP, and G6P. We then generated HKIII constructs with mutations in these putative binding sites (Figure 2A). Histidine-tagged wildtype and mutant proteins were expressed in a bacterial system and purified on a nickel column, and the Figure 2. Mutations in putative N-terminal substrate-binding residues of HKIII alter catalytic activity in the C-terminal domain. (A) Six mutant HKIII proteins were generated, each containing a mutation in one of the N-terminal or C-terminal binding sites for G6P, ATP, or glucose. The catalytic activity of each mutant protein was assessed via the G6P/NADPH-coupling assay. (B) Mutations in the putative N-terminal G6P- and glucose-binding sites significantly impaired HKIII specific activity. *P,0.05 vs WT. Vmax = .136.010; .096.009; .126.009; .0986.009 for Wt, E95A, S102A, and T174A respectively. There was no detectable change in absorbance for the C-terminal mutant constructs. *P,0.05 v. Wt; Data are presented as mean6SEM, N$3 replicates for each construct. doi:10.1371/journal.pone.0013823.g002 PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13823