Mandy Kwong

Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells.

The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119–myristate complex, we identified highly conserved phenylalanines within a hydrophobic β sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.