Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4K␤ (PI4KIII␤) and type II PI4K␣ (PI4KII␣). Here we report that PI4KIII␤ and PI4KII␣ localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIII␤ was enriched in early Golgi compartments, whereas PI4KII␣ colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIII␤ and PI4KII␣ or in vitro by the PI4KIII␤ inhibitor wortmannin. Our data suggest that both PI4KIII␤ and PI4KII␣ contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.

The mechanisms that regulate endoplasmic reticulum (ER) exit-site (ERES) assembly and COPII-mediated ER export are currently unknown. We analyzed the role of phosphatidylinositols (PtdIns) in regulating ER export. Utilizing pleckstrin homology domains and a PtdIns phosphatase to specifically sequester or reduce phosphorylated PtdIns levels, we found that PtdIns 4-phosphate (PtsIns4P) is required to promote COPII-mediated ER export. Biochemical and morphological in vitro analysis revealed dynamic and localized PtsIns4P formation at ERES. PtdIns4P was utilized to support Sar1-induced proliferation and constriction of ERES membranes. PtdIns4P also assisted in Sar1-induced COPII nucleation at ERES. Therefore, localized dynamic remodeling of PtdIns marks ERES membranes to regulate COPII-mediated ER export.

Here we present evidence that the epithelial sodium channel (ENaC), a heteromeric membrane protein whose surface expression is regulated by ubiquitination, is present in clathrin-coated vesicles in epithelial cells that natively express ENaC. The channel subunits are ubiquitinated and co-immunoprecipitate with both epsin and clathrin adaptor proteins, and epsin, as expected, co-immunoprecipitates with clathrin adaptor proteins. The functional significance of these interactions was evaluated in a Xenopus oocyte expression system where co-expression of epsin and ENaC resulted in a downregulation of ENaC activity; conversely, co-expression of epsin subdomains acted as dominant-negative effectors and stimulated ENaC activity. These results identify epsin as an accessory protein linking ENaC to the clathrin-based endocytic machinery thereby regulating the activity of this ion channel at the cell surface.

The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential -mucolipin-1 (TRP-ML1) ion channel. The " biogenesis " model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid traffi cking delays in MLIV patient fi broblasts. Because membrane traffi c aberrations may be secondary to lipid buildup in chronically TRP-ML1 -defi cient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffi c. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 defi ciency on membrane traffi c. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1 -defi cient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confi rmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fi broblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV.

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...