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The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number ("'150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.A wide variety of molecules gain entry into cells by receptormediated endocytosis (1,2). Diverse ligands, including low density lipoproteins, asialoglycoproteins, epidermal growth factor, a2 macroglobulin, lysosomal enzymes, and certain hormones, toxins, and viruses traverse strikingly similar, if not identical, pathways. Binding to specific cell-surface receptors is followed by internalization involving specialized regions of the plasma membrane, the coated pits. Between their internalization and their ultimate fate, which is often lysosomal degradation, appropriately tagged ligands are visible by electron microscopy within a number of morphologically varied intracellular vesicles. We have examined this pathway for the receptor-mediated endocytosis of asialoglycoproteins in hepatocytes and have shown that the receptor enters the cell as a complex with its ligand, whereupon the latter subsequently dissociates and is degraded (3, 4). In contrast to the ligand, the receptor is reutilized (5, 6). We have presented evidence that the intracellular dissociation of asialoglycoproteins is facilitated by encounter with an acidic environment prior to delivery to lysosomes (4, 7), a step that we believe to be critical in receptor reutilization. There is growing evidence that a number of other ligands are similarly exposed to nonlysosomal environments of low pH in their movement through the cell (8-11). This suggests that an acidic endocytic vesicle may participate in the intracellular transit of many ligands. As with asialoglycoproteins, a number of ligands interact with their respective receptors in a highly pH-dependent fashion, and many of these receptors have been shown to be reutilized (1).A notable exception to this generalized picture of receptormediated endocytosis involves the iron-binding protein transferrin (reviewed in ref. 12). Diferric transferrin enters the cell bound to its specific receptor, but internalization of the receptor-bound transferrin does not result in transferrin degradation (13)(14)(15). Instead, both the receptor and the apoprotein ligand are returned to the cell's exterior with retention of iron within the cell. Although coated pits (16) and an acidic, nonlysosomal microenvironment (9) have been shown to be involved in the transfe...

O-linked N-acetylglucosamine (O-GlcNAc) is an evolutionarily conserved modification of nuclear pore proteins, signaling kinases, and transcription factors. The O-GlcNAc transferase (OGT) catalyzing O-GlcNAc addition is essential in mammals and mediates the last step in a nutrient-sensing ''hexosamine-signaling pathway.'' This pathway may be deregulated in diabetes and neurodegenerative disease. To examine the function of O-GlcNAc in a genetically amenable organism, we describe a putative null allele of OGT in Caenorhabditis elegans that is viable and fertile. We demonstrate that, whereas nuclear pore proteins of the homozygous deletion strain are devoid of O-GlcNAc, nuclear transport of transcription factors appears normal. However, the OGT mutant exhibits striking metabolic changes manifested in a Ϸ3-fold elevation in trehalose levels and glycogen stores with a concomitant Ϸ3-fold decrease in triglycerides levels. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity, and dauer formation. The OGT knockout suppresses dauer larvae formation induced by a temperature-sensitive allele of the insulin-like receptor gene daf-2. Our findings demonstrate that OGT modulates macronutrient storage and dauer formation in C. elegans, providing a unique genetic model for examining the role of O-GlcNAc in cellular signaling and insulin resistance.is a nucleocytoplasmic modification present throughout eukaryotic evolution with the possible exception of yeast (1, 2). Although many intracellular proteins such as nuclear pore components and transcription factors bear O-GlcNAc, the precise function of the modification is unknown. Evidence in mammals suggests a role for O-GlcNAc in the development of insulin resistance associated with noninsulindependent diabetes mellitus (3, 4). A number of lines of evidence also link O-GlcNAc to transcriptional regulation and neurodegeneration (1, 2). O-GlcNAc addition is partly driven by the levels of UDP-GlcNAc derived from the hexosamine biosynthetic pathway. This pathway is a nutrient-sensing pathway implicated in cellular signaling (1, 2). The uncertainty regarding the precise function of O-GlcNAc is perhaps to be expected given the many substrates modified by this glycan addition. Furthermore, O-GlcNAc is a dynamic modification; the levels of O-GlcNAc are maintained by the action of a glycosytransferase [O-linked GlcNAc transferase (OGT)] and a hexosaminidase (O-GlcNAcase) (1, 2). Mammalian O-GlcNAcase exists as two splice variants and is relatively specific for nucleocytoplasmic O-GlcNAc (1, 5, 6). The transferase, OGT, catalyzes the transfer of O-GlcNAc to Ser͞Thr residues. This enzyme has been identified from a number of sources, including plants, human, rat, mouse, and the nematode Caenorhabditis elegans (7,8). In plants, the OGT homolog Spindly is involved in plant-signaling pathways (9, 10). These evolutionarily conserved proteins share a similar overall structure; OGT is composed of multiple protein domains, including tetratricopepti...