The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X L, BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX-and BAK-independent role for a BCL2-related protein in development.autophagy ͉ mitochondria ͉ BCL2 family B CL2-related proteins play essential roles in the regulation of programmed cell death. Members of the BCL2 family are divided into subgroups based on the presence of one or more BCL2 homology domains (BH1-BH4) (1). BCL2-related proteins possessing a single BH3 domain (BH3-only proteins) are activated by diverse death-inducing stimuli including DNA damage, glucocorticoids, and growth factor deprivation, and their signals are integrated at the mitochondria by the multidomain proapoptotic proteins BAX and BAK (2). BH3-only proteins activate BAX and BAK either directly or indirectly, through binding to and inhibiting the function of antiapoptotic BCL2-related proteins (3-5). BAX or BAK activation in turn causes cytochrome c release, caspase activation, and apoptosis (6, 7). BNIP3 and NIX (also known as BNIP3L) are related proteins with limited homology to BH3-only proteins in a BH3-like domain (8-10). BNIP3 and NIX have uncertain biological function. BNIP3 and NIX localize to the mitochondria when overexpressed, induce cytochrome c release, and cause apoptosis (11-13), however, BNIP3 also causes necrosis-like cell death (14). Hypoxia induces and retinoblastoma protein represses BNIP3 expression through HIF-1␣ and E2F binding sites in the BNIP3 promoter, respectively (15-17). In contrast, NIX is induced by G q -coupled hypertrophic agonists in neonatal rat cardiomyocytes, by p53 in U2OS osteosarcoma cells, and by differentiation of human erythroid cells (18)(19)(20). Accordingly, NIX functions as an effector of G q -dependent cardiomyopathy and negatively regulates tumor growth in nude mice injected with U2OS osteosarcoma cells (19,21). NIX has a role in erythroid development, because Nix Ϫ/Ϫ mice exhibit anemia and erythroid hyperplasia (22). Results and DiscussionDefective Erythropoiesis in Nix ؊...

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O 2 , CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20 -500-fold increases in the rates of O 2 dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O 2 in both the ␣ and ␤ chains (⌬G His(E7)H-bond ≈ ؊8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k O2 , In 1970, Perutz (1) proposed that the distal histidines located at the E7 helical positions, 3 ␣His-58 and ␤His-63, play crucial structural roles for regulating both the affinities and rates of O 2 binding to adult human hemoglobin (HbA). 4 These ideas were based on the suggestion by Pauling (2) that His(E7) could stabilize bound O 2 by donating a hydrogen bond to the partial negative charge on the superoxide-Fe(III)-like FeO 2 complex and on the idea by Perutz and Mathews (3) that the distal histidine could also be acting as gate for ligand entry and exit. Studies of model heme compounds and naturally occurring globins with His(E7) replacements suggested strongly that the distal histidine also plays a key role in discrimination between O 2 and CO binding (4 -9).The first mutagenesis studies on sperm whale Mb and human HbA reported that His(E7) to Gly mutations in ␣ subunits and Mb cause marked increases in the rates of O 2 dissociation and significant decreases in affinity, both of which indicate strong stabilization of the FeO 2 complex by proton donation from the wild-type His(E7) side chain (7, 10, 11). In addition, the association rate constants for O 2 and CO binding increased 5-10-fold. The latter results were interpreted in terms of the distal histidine gate mechanism, with the His(E7) to Gly mutation resulting in an open E7 channel. In contrast, neither the dissociation nor association rate constants for O 2 binding to the R state ␤Gly(E7) mutant of HbA appeared to increase significantly, implying no electrostatic stabilization of bound ligands by the native His(E7) in ␤ subunits and an already open gate or alternative pathway. These surprising kinetic results were explained by the first high resolution structure of human oxyhemoglobin published by Shaanan (12), in which the N⑀H atoms of distal histidine in ␤ subunits seemed to be further away from the bound O 2 atoms and pointing toward the heme plane.Between 1989 and 1999, our group and others constructed large libraries of mammalian Mb mutants as a model system for understanding the structural mechanisms of ligand binding to vertebrate globins involved in O 2 transport and storage. A detailed molecular mechanism for O 2 binding has emerged. Ligand entry into myoglobin occurs through t...

Alkyl isocyanides (CNRs) identify pathways for diatomic ligand movement into and out of Mb, with their side chains acting as transition state analogs. The bound alkyl groups point either into the back of the distal pocket (in conformation, νCN ≈ 2070–2090 cm−1), which allows hydrogen bond donation from His64(E7) to the isocyano group, or toward solvent through an open His(E7) channel (out conformation, νCN ≈ 2110–2130 cm−1), which prevents polar interactions with the isocyano atoms. Fractions of the in conformer (Fin) were measured by FTIR spectroscopy for methyl through n-pentyl isocyanide bound to a series of 20 different distal pocket mutants of sperm whale myoglobin and found to be governed by the ease of rotation of the His(E7) side chain, distal pocket volume and steric interactions, and, for the longer isocyanides, the unfavorable hydrophobic effect of placing their terminal carbon atoms into the solvent phase in the out conformation. There are strong correlations between the fraction of in conformer, Fin, for long chain MbCNR complexes measured by FTIR spectroscopy, the fraction of geminate recombination of photodissociated O2, and the bimolecular rates of O2 entry into the distal pocket. These correlations indicate that alkyl isocyanides serve as transition state analogs for the movement of O2 into and out of the binding pocket of Mb.

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virusinduced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation. IntroductionFriend disease is a multistage viral disease in mice. [1][2][3] In the initial stage of Friend disease, expression of the viral oncoprotein, gp55, causes uncontrolled erythroid proliferation and erythroblastosis. Erythroblastosis is a condition characterized by the rapid accumulation of immature erythroid cells leading to acute splenic enlargement. In the later stages of Friend disease, retroviral integrations in Sfpi1, p53, and Nfe2 cause progression to erythroleukemia. Friend virus is a complex of 2 viruses, Friend murine leukemia virus and spleen focus-forming virus (SFFV). 4,5 SFFV encodes a mutant envelope protein, gp55, which is necessary and sufficient for the erythroblastosis stage of the disease. 6 There are 2 strains of Friend virus, an anemia-inducing strain (FVA) and a polycythemiainducing strain (FVP). 1,7 The amino acids responsible for this phenotypic difference have been localized to the transmembrane domain of gp55 (gp55 A and gp55 P ). 8 Previously, it was shown that gp55 P could interact with the erythropoietin receptor (EPOR) and support proliferation of the interleukin-3 (IL-3)-dependent cell line Ba/F3. 9 Based on this observation, it was proposed that Friend virus causes erythroblastosis through constitutive activation of the EPOR.Additional insights into the mechanism of action of Friend virus have been provided by host factors that confer resistance or susceptibility to Friend disease. Fv1 and Fv4 confer resistance to Friend disease through interference with the virus life cycle or interference with virus binding to the ecotropic receptor, respectively. 10,11 Fv2 confers susceptibility t...