Considerable advances have been made in recent years in our understanding of the biochemistry of mucin-type glycoproteins. This class of compounds is characterized mainly by a high level of O-linked oligosaccharides. Initially, the glycoproteins were solely known as the major constituents of mucus. Recent studies have shown that mucins from the gastrointestinal tract, lungs, salivary glands, sweat glands, breast, and tumor cells are structurally related to high-molecular-weight glycoproteins, which are produced by epithelial cells as membrane proteins. During mucin synthesis, an orchestrated sequence of events results in giant molecules of Mr 4 to 6 x 10(6), which are stored in mucous granules until secretion. Once secreted, mucin forms a barrier, not only to protect the delicate epithelial cells against the extracellular environment, but also to select substances for binding and uptake by these epithelia. This review is designed to critically examine relations between structure and function of the different compounds categorized as mucin glycoproteins.

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In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron miscroscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes. INTRODUCTIONThe best-documented way of endocytosis is receptor-mediated uptake of ligands via clathrin-coated vesicles (reviewed in Schmid, 1997). Receptors are recruited and concentrated into clathrin-coated pits at the plasma membrane. After coated vesicle formation, the clathrin coat is removed by the concerted action of auxilin and heat shock protein 70 (Ungewickell et al., 1995). The uncoated vesicles fuse with early endosomes (EEs) in a rab5-regulated manner (Rubino et al., 2000). The highly dynamic EE consists of a vacuolar part (also known as sorting endosome) and emerging tubular extensions. The tubulovacuolar organization of EEs reflects their critical role in protein sorting. Receptors destined for degradation in lysosomes, such as epidermal growth factor receptor (EGFR) and growth hormone receptor (GHR), are incorporated into small vesicles in the lumen of the endosomal vacuole, which form by inward budding of the limiting membrane (microautophagy). Recycling receptors, such as the transferrin receptor (TfR) enter the tubular extensions of EEs and recycling endosomes (REs) from where they are routed back to the plasma membrane (Stoorvogel et al., 1987;Hopkins et al., 1994).Despite the progress on the structural and molecular characterization of EEs, little is known about the mechanisms of intraendosomal protein sorting. It has been suggested that sorting occurs in a process of "iterative fractioning" (Dunn et al., 1989). This model, based on recycling receptors of which the ligands are released upon entry in the acidic environment of the sorting endosome, proposes that receptors rapidly and continuously enter the tubular extensions for recycling to the plasma membrane, whereas ...

β-catenin mediates Wnt signaling by acting as the essential co-activator for TCF transcription factors. Wnt signaling increases the half-life and therefore the absolute level of β-catenin in responding cells. The current model states that these changes in β-catenin stability set the threshold for Wnt signaling. However, we find that pharmacological inhibition of proteasome activity by ALLN leads to accumulation of cytosolic β-catenin but not to increased TCF-mediated transcription. In addition, in temperature-sensitive ubiquitylation mutant CHO cells inhibition of ubiquitylation increases β-catenin levels, but does not induce transcriptional activation of TCF reporter genes. Using an antibody specific for β-catenin dephosphorylated at residues Ser37 and Thr41, we show that Wnt signals specifically increase the levels of dephosphorylated β-catenin, whereas ALLN does not. We conclude that changes in the phosphorylation status of the N-terminus of β-catenin that occur upon Wnt signaling independently affect the signaling properties and half-life of β-catenin. Hence, Wnt signals are transduced via N-terminally dephosphorylated β-catenin.

Corresponding authorThe low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor with the ability to bind and endocytose several structurally and functionally distinct ligands. A 39 kDa receptorassociated protein (RAP) inhibits all ligand interactions with LRP in vitro. In the present study, we demonstrate that RAP is an endoplasmic reticulum (ER) resident protein. The tetrapepetide sequence HNEL at the C-terminus of RAP is both necessary and sufficient for RAP retention within the ER. Metabolic labeling combined with cross-linking studies shows that RAP interacts with LRP in vivo. Pulse-chase analysis reveals that this association is transient early in the secretory pathway and coincides with LRP aggregation and reduced ligand binding activity. Both internal triplicated LRP binding domains on RAP and multiple RAP binding domains on LRP appear to contribute to the aggregation of LRP and RAP. Dissociation of RAP from LRP results from the lower pH encountered later in the secretory pathway and correlates with an increase in LRP ligand binding activity. Taken together, our results thus suggest that RAP functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity along the secretory pathway.

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In eukaryotic cells, secretory proteins and glycoproteins migrate from the rough endoplasmic reticulum, their site of synthesis, through Golgi vesicles before being released from the cell. Cellular and viral integral plasma membrane glycoproteins are co-translationally inserted into the rough endoplasmic reticulum membrane and follow a similar pathway to the cell surface. Previous studies using endoglycosidase H (Endo H) suggested that in rat hepatoma cells the vesicular stomatitis virus (VSV) G protein, albumin and transferrin migrate from the rough endoplasmic reticulum to the Golgi apparatus at different rates. Here we show directly that in human hepatoma HepG2 cells, five secreted proteins mature from the rough endoplasmic reticulum to Golgi vesicles at characteristic rates which differ at least threefold. The results are incompatible with bulk-phase movement of the luminal contents of the endoplasmic reticulum, and suggest that there is a membrane-bound receptor that selectively mediates the transport of secretory proteins from the rough endoplasmic reticulum to the Golgi.

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.