We have examined the traf®cking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immuno¯uores-cence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXF and was localized primarily to lysosomes at steady state, signi®cant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and ef®ciently transported to lysosomes. Analysis of endolyn±CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells.

The sialomucin endolyn is a transmembrane protein with a unique trafficking pattern in polarized Madin-Darby canine kidney cells. Despite the presence of a cytoplasmic tyrosine motif that, in isolation, is sufficient to mediate basolateral sorting of a reporter protein, endolyn predominantly traverses the apical surface en route to lysosomes. Apical delivery of endolyn is disrupted in tunicamycin-treated cells, implicating a role for N-glycosylation in apical sorting. Site-directed mutagenesis of endolyn's eight N-glycosylation sites was used to identify two N-glycans that seem to be the major determinants for efficient apical sorting of the protein. In addition, apical delivery of endolyn was disrupted when terminal processing of N-glycans was blocked using glycosidase inhibitors. Missorting of endolyn occurred independently of the presence or absence of the basolateral sorting signal, because apical delivery was also inhibited by tunicamycin when the cytoplasmic tyrosine motif was mutated. However, we found that apical secretion of a soluble mutant of endolyn was N-glycan independent, as was delivery of glycosylphosphatidylinositol-anchored endolyn. Thus, specific N-glycans are only essential for the apical sorting of transmembrane endolyn, suggesting fundamental differences in the mechanisms by which soluble, glycosylphosphatidylinositol-anchored, and transmembrane proteins are sorted. INTRODUCTIONProper functioning of polarized epithelial cells necessitates the maintenance of differentiated apical and basolateral plasma membranes, which requires appropriate sorting of newly synthesized proteins to these distinct domains. In the biosynthetic pathway, newly synthesized apical and basolateral proteins are sorted upon reaching the trans-Golgi network (TGN). Basolateral sorting signals, including some tyrosine-containing tetrapeptide motifs and di-leucine motifs, are generally localized to the cytoplasmically exposed portions of these proteins (Nelson and Yeaman, 2001). These signals are thought to bind directly to adaptor protein complexes, which mediate sorting of the respective transmembrane proteins into basolaterally directed transport vesicles, analogous to the sorting of plasma membrane proteins into endocytic vesicles by AP-2 (Folsch et al., 2001;Simmen et al., 2002;Bonifacino and Traub, 2003). By contrast, apical sorting signals are less well defined and reside frequently within the membrane-or lumenally oriented regions of these molecules (Nelson and Yeaman, 2001). Membrane-embedded signals include glycosylphosphatidylinositol (GPI)-anchors attached to the carboxy terminus of some proteins and amino acid sequences within the transmembrane domains of other apical proteins. It has been postulated that association with glycolipid-enriched lipid microdomains may play a role in the apical delivery of these proteins (Ikonen and Simons, 1998;Paladino et al., 2002). In addition, both N-and O-linked glycosylation have been demonstrated to be necessary for the correct apical delivery of several proteins (Scheiffe...

Mucolipin-1 (ML1) is a member of the transient receptor potential ion channel superfamily that is thought to function in the biogenesis of lysosomes. Mutations in ML1 result in mucolipidosis type IV, a lysosomal storage disease characterized by the intracellular accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, and mucopolysaccharides. Little is known about how ML1 trafficking or activity is regulated. Here we have examined the processing and trafficking of ML1 in a variety of cell types. We find that a significant fraction of ML1 undergoes cell type-independent cleavage within the first extracellular loop of the protein during a late step in its biosynthetic delivery. To determine the trafficking route of ML1, we systematically examined the effect of ablating adaptor protein complexes on the localization of this protein. Whereas ML1 trafficking was not apparently affected in fibroblasts from mocha mice that lack functional adaptor protein complex (AP)-3, small interfering RNA-mediated knockdown revealed a requirement for AP-1 in Golgi export of ML1. Knockdown of functional AP-2 had no effect on ML1 localization. Interestingly, cleavage of ML1 was not compromised in AP-1-deficient cells, suggesting that proteolysis occurs in a prelysosomal compartment, possibly the trans-Golgi network. Our results suggest that posttranslational processing of ML1 is more complex than previously described and that this protein is delivered to lysosomes primarily via an AP-1-dependent route that does not involve passage via the cell surface.

Phosphatidylinositols (PI) play important roles in regulating numerous cellular processes including cytoskeletal organization and membrane trafficking. The control of PI metabolism by phosphatidylinositol kinases has been the subject of extensive investigation; however, little is known about how phosphatidylinositol kinases regulate traffic in polarized epithelial cells. Because phosphatidylinositol 4-kinase (PI4K)-mediated phosphatidylinositol 4-phosphate (PI(4)P) production has been suggested to regulate biosynthetic traffic in yeast and mammalian cells, we have examined the role of PI4K␤ in protein delivery in polarized MDCK cells, at different levels of the biosynthetic pathway. Expression of wild type PI4K␤ had no effect on the rate of transport of influenza hemagglutinin (HA) through the Golgi complex, but inhibited the rate of trans-Golgi network (TGN)-to-cell surface delivery of this protein. By contrast, expression of dominant-negative, kinase-dead PI4K␤ (PI4K␤ D656A ) inhibited intra-Golgi transport but stimulated TGN-to-cell surface delivery of HA. Moreover, expression of PI4K␤ D656A significantly increased the solubility in cold Triton X-100 of HA staged in the TGN, suggesting that altered association of HA with lipid rafts may be responsible for the enhanced transport rate. Both wild type and kinase-dead PI4K␤ inhibited basolateral delivery of vesicular stomatitis virus G protein, suggesting an effector function for PI4K␤ in the regulation of basolateral traffic. Thus, by contrast with the observed requirement for PI4K␤ activity and PI(4)P for efficient transport in yeast, our data suggest that changes in PI(4)P levels can stimulate and inhibit Golgi to cell surface delivery in mammalian cells.

The mechanisms by which polarized epithelial cells target distinct carriers enriched in newly synthesized proteins to the apical or basolateral membrane remain largely unknown. Here we investigated the effect of phosphatidylinositol metabolism and modulation of the actin cytoskeleton, two regulatory mechanisms that have individually been suggested to function in biosynthetic traffic, on polarized traffic in Madin-Darby canine kidney cells. Overexpression of phosphatidylinositol 5-kinase (PI5K) increased actin comet frequency in Madin-Darby canine kidney cells and concomitantly stimulated trans-Golgi network (TGN) to apical membrane delivery of the raft-associated protein influenza hemagglutinin (HA), but did not affect delivery of a non-raft-associated apical protein or a basolateral marker. Modulation of actin comet formation by pharmacologic means, by overexpression of the TGN-localized inositol polyphosphate 5-phosphatase Ocrl, or by blockade of Arp2/3 function had parallel effects on the rate of apical delivery of HA. Moreover, HA released from a TGN block was colocalized in transport carriers in association with PI5K and actin comets. Inhibition of Arp2/3 function in combination with microtubule depolymerization led to a virtual block in HA delivery, suggesting synergistic coordination of these cytoskeletal assemblies in membrane transport. Our results suggest a previously unidentified role for actin comet-mediated propulsion in the biosynthetic delivery of a subset of apical proteins.