Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

No abstract

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 ؊...

Two donor-acceptor (D-A) copolymer PDVTs based on diketopyrrolopyrole and (E)-2-(2-(thiophen-2-yl)vinyl)thiophene (TVT) units are synthesized for solution-processed field-effect transistors (FETs). The highly π-extended TVT units strengthen the coplanarity of the polymer backbone. FETs based on PDVTs show high mobilities above 2.0 cm(2) V(-1) s(-1) with a current on/off ratio of 10(5)-10(7) , high shelf storage, and operation stability.

BNIP3 and NIX are proteins related to the BH3-only family, which induce both cell death and autophagy. Consistent with their ability to induce cell death, BNIP3 and NIX are implicated in the pathogenesis of cancer and heart disease. In tumor cells, BNIP3 and NIX are regulated by hypoxia, and the deregulation of BNIP3 or NIX expression is associated with tumor growth. In heart muscle, BNIP3 and NIX are regulated by hypoxia and Gaq-dependent signaling, respectively, and their expression is associated with decreased myocardial function. Apart from their role in cell death, BNIP3 and NIX are also implicated in the induction of autophagy. In erythroid cells, NIX is required for a specialized type of autophagy that targets mitochondria for elimination (mitophagy). Similarly, BNIP3 regulates mitophagy in response to hypoxia. In this review, we will discuss possible mechanisms by which BNIP3 and NIX induce cell death and mitophagy. We will also consider the potential relationship between cell death pathways and autophagy in development and homeostasis. BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3) and BNIP3-like (BNIP3L), also known as NIX, are proteins with homology to BCL2 in the BH3 domain, which induce both cell death and autophagy. Although other proteins may induce cell death or autophagy more efficiently than BNIP3 or NIX, the ability of these proteins to do both makes them useful reagents to explore the relationship between these alternate cell fates. With this in mind, here we review the role of BNIP3 and NIX in cell death and autophagy, and discuss potential mechanisms by which they may function. In addition, we discuss a specialized type of autophagy that is relevant during development, namely mitophagy, in the erythroid lineage. Discovery and Characterization of BNIP3BNIP1, BNIP2, and BNIP3, were initially identified in a yeast two-hybrid screen as BCL2 and adenovirus E1B 19-kDainteracting proteins.

Production of a red blood cell's hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy. IntroductionErythroid differentiation involves progression through morphologically distinct nucleated precursor stages, from proerythroblast to orthochromatic erythroblasts, prior to enucleation and maturation of the nascent reticulocyte. This process results in a successive reduction in cell volume, massive increase in hemoglobin production and conversion to a purely glycolytic pathway for energy production. Unlike most cells, mature red blood cells have no nucleus or organelles. Following enucleation of orthochromatic erythroblasts, nascent reticulocytes mature over the course of 48 to 72 hours and are cleared of all intracellular organelles, including mitochondria and ribosomes. Approximately 30% of red cell hemoglobin is produced in reticulocytes, and since heme is synthesized in the mitochondria these organelles are among the last to be eliminated (reviewed in Geminard et al 1 ). The programmed clearance of mitochondria that occurs in reticulocytes makes it an ideal system for studying the molecular pathways involved in mitochondrial degradation. Understanding the process by which cells degrade mitochondria is important not only for red cell maturation, but also for other cell types as accumulation of damaged or dysfunctional mitochondria has been implicated in aging and various pathologic processes such as diabetes, cancer, neurodegeneration, and ␣1 antitrypsin disease (reviewed in Pieczenik and Neustadt 2 ).The molecular pathways involved in mitochondrial degradation in reticulocytes are unknown, although several important insights have been made into this process. 15-Lipoxygenase is a lipid-peroxidizing enzyme whose expression peaks in reticulocytes shortly before organelle degradation. 3 It integrates into organelle membranes, disrupts mitochondrial membrane integrity allowing release of proteins from the organelle lumen and proteasome-dependent degradation of lumenal and integral membrane proteins. 4,5 Chemical inh...

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.gremlin ͉ expression profiling ͉ microarray ͉ crypt maturation program ͉ myofibroblast

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.