along with continued work to generate a crystal structure for HKIII will help clarify the effects of N-terminal substrate binding on HKIII activity. The protective effects of HKI and HKII are believed to occur through changes in the glucose-phosphorylation and mitochon- drial-binding activity of the enzymes [27]. Since HKIII does not bind to mitochondria, we proposed that its protective effects are due to: 1) an increase in glucose phosphorylation, energy production and mitochondrial biogenesis, and 2) a reduction in cellular ROS levels. Our results demonstrate that overexpression of HKIII results in an increase in cellular ATP levels and mitochondrial biogenesis, and a decrease in oxidant induced ROS production. Although these results do not demonstrate a cause and effect relationship between these factors and cell death, they implicate their possible role in this process. The binding of HKI and HKII to the mitochondria is physiologically significant, because it contributes to their cytopro- tective effects, and dislocation of HKI and HKII to the cytoplasm is associated with higher rates of cell death [26,27,44]. Mitochon- drial binding occurs through a 21-amino-acid, N-terminal sequence encoded by exon 1 of the HKI and HKII genes, which is predicted to form a hydrophobic alpha-helix that binds to the outer membrane of the mitochondria. In this study, we investigated whether the protective effects of HKIII could be enhanced by replacing the N-terminal 32 amino acids of HKIII (encoded by exon 1b) with the 21-amino-acid mitochondrial- targeting sequence of HKII (encoded by exon 1). Our results indicate that the sequence in HKIII, encoded by exon 1b, is critical for protein folding, since its deletion or its replacement with the mitochondrial-targeting sequence of HKII resulted in the formation of intracellular aggregates. The sequences encoded by exon 1 are unique to the 100-kDa mammalian HKs and they are not present in the 50-kDa HKs expressed in yeast and other organisms. In 100-kDa enzymes, the N-half (encoded by exons 2 through half of exon 10) and the C-half (encoded by exons 10-18) form the catalytic and regulatory portion of the proteins, while exon 1 is not part of the duplication process and encodes unique sequences. We showed that unlike HKI and HKII, this region of HKIII is needed for proper folding of the protein and that its deletion makes the enzyme insoluble, leading to aggregate formation. In summary, the results reported here are the first to describe the regulation of HKIII expression and activity and to characterize the role of HKIII in cytoprotection. HKIII expression is regulated by hypoxia through a HIF-dependent signal transduction pathway, and the activity of HKIII is also regulated at the protein level by the influence of N-terminal substrate binding on C- terminal catalysis. HKIII overexpression promotes cell survival in response to oxidative stress, perhaps by increasing cellular ATP levels, decreasing ROS production, and promoting mitochondrial biogenesis, and the sequence encoded by exon 1b of HKIII is critical for proper protein folding, whereas the corresponding sequences in HKI and HKII function as mitochondrial-targeting signals. Collectively, these observations provide insights into the specific role of the HKIII isozyme in cell metabolism and function. Supporting Information Table S1 Primer Sequences used for PCR Amplification Found at: doi:10.1371/journal.pone.0013823.s001 (0.60 MB TIF) Figure S1 HKIII levels do not change in N1S1 cells after cAMP or AMPK activation. HKIII mRNA expression was measured in N1S1 cells cultured for 24 hours with or without (A) 5 microM forskolin (an inducer of cAMP production) or (B) 1 mM AICAR HK3 Regulation and Protection (an activator of AMPK). HKIII mRNA levels in treated and untreated cells were similar. Data are presented as mean+SEM, N = 3 for all groups. Found at: doi:10.1371/journal.pone.0013823.s002 (0.52 MB TIF) Figure S2 Hypoxia regulates HKII expression in N1S1 cells. (A) HKII mRNA levels were measured in N1S1 cells cultured under normoxic or hypoxic (1.5% O2) conditions for 24 hours. HKII levels were significantly increased after hypoxia treatment. (B) N1S1 cells were cultured under normoxic conditions for 24 hours with or without a HIF stabilizer (DMOG, 100 and 300 mM). HKII mRNA levels increased significantly after DMOG treatment. (C) Comparative mRNA expression levels of the HKII and HKIII isoforms in N1S1 cells cultured under normoxic or hypoxic (1.5% O2) conditions for 24 hours, demonstrating the higher levels of HKII expression in this cell type. *P,0.05 v. control; Data are presented as mean6SEM, N = 3–5 for all groups. Found at: doi:10.1371/journal.pone.0013823.s003 (0.45 MB TIF) Figure S3 Overexpression of GFP and HKIII-GFP in HEK cells labeled with Mitosox. (A) GFP fluorescent overlay for the HEK cell images displayed in figure 5B, demonstrating the overexpression of the GFP and HKIII-GFP constructs in cells treated with or without 500 mM H2O2 (3 h), and then incubated with (MitoSox). Cell nuclei are labeled with Hoechst (blue). Found at: doi:10.1371/journal.pone.0013823.s004 (2.61 MB TIF) Figure S4 Overexpression of GFP and HKIII-GFP in HEK cells labeled with TMRE. (A) GFP fluorescent overlay for the HEK cell images displayed in figure 6 A-D, demonstrating the overexpression of the GFP (top panels) and HKIII-GFP (bottom panels) constructs in cells loaded with TMRE(orange) prior to the treatment condition indicated. Cell nuclei are labeled with Hoechst (blue). Found at: doi:10.1371/journal.pone.0013823.s005 (5.19 MB TIF) Figure S5 Overexpression of the N and C-terminal domains of HKIII in HEK293 cells. (A) Plasmid constructs coding for the expression of the N and C-terminal domains of HKIII. Each construct also coded for GFP expression at the C-terminus. (B) Live cell confocal images of HEK cells after transfection with the indicated constructs reveals soluble expression of the N-terminal half of HKIII (NHKIII) similar to the full length HKIII construct, while overexpression of the C-terminal domain resulted in aggregate formation. The cells were incubated with TMRE (red) to identify mitochondria, and nuclei were labeled with Hoechst (blue). Found at: doi:10.1371/journal.pone.0013823.s006 (2.89 MB TIF) Figure S6 Progressive deletion of 8-amino-acid segments from the N-terminus of HKIII do not cause protein aggregation. (A) Plasmids expressing truncated versions of HKIII that lacked the first 8 (HKIII-8), 16 (HKIII-16), or 24 (HKIII-24) amino acids of the N-terminal sequence encoded by exon 1b were constructed and expressed in HEK293 cells; each construct also coded for GFP expression at the C-terminus. (B) Live cell confocal images of HEK cells after transfection with the indicated constructs showed that the full-length protein and each of the three serial truncation constructs were expressed in the cytoplasm and did not form cellular aggregates. The cells were incubated with TMRE (red) to identify mitochondria, and nuclei were label with Hoechst (blue). Found at: doi:10.1371/journal.pone.0013823.s007 (3.35 MB TIF) Figure S7 Western blot for protein expression for the HKIII constructs. (A) Western blot of cell lysates from HEK293 cells overexpressing the indicated constructs. For each construct, 1.5 mg PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e13823