Kelly M. Weixel

Distinct Golgi Populations of Phosphatidylinositol 4-Phosphate Regulated by Phosphatidylinositol 4-Kinases

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 PI4Kβ (PI4KIIIβ) and type II PI4Kα (PI4KIIα). Here we report that PI4KIIIβ and PI4KIIα localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIβ was enriched in early Golgi compartments, whereas PI4KIIα 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 PI4KIIIβ and PI4KIIα or in vitro by the PI4KIIIβ inhibitor wortmannin. Our data suggest that both PI4KIIIβ and PI4KIIα 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.

Clathrin-mediated Endocytosis of the Epithelial Sodium Channel ROLE OF EPSIN

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 down-regulation of ENaC activity; conversely, co-expression of epsin sub-domains 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.

Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis

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 trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1–deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. 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 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1–deficient 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 confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. 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.

Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1.

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.

Phosphatidylinositol 5-Kinase Stimulates Apical Biosynthetic Delivery via an Arp2/3-dependent Mechanism

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.

Recycling of MUC1 Is Dependent on Its Palmitoylation

MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXφ motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.

A Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Generates a Novel Internalization Sequence and Enhances Endocytic Rates

Cystic fibrosis is a common lethal genetic disease among Caucasians. The cystic fibrosis gene encodes a cyclic adenosine monophosphate-activated chloride channel (cystic fibrosis transmembrane conductance regulator (CFTR)) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. Mutations in CFTR fall into two broad categories; those that affect protein biosynthesis/stability and traffic to the cell surface and those that cause altered channel kinetics in proteins that reach the cell surface. Here we report a novel mechanism by which mutations in CFTR give rise to disease. N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. The sole consequence of this novel mutation is to generate a novel tyrosine-based endocytic sequence within an intracellular loop in CFTR leading to increased removal from the cell surface and a reduction in the steady-state level of CFTR at the cell surface.

Phosphatidylinositol 4-Phosphate 5-Kinase Reduces Cell Surface Expression of the Epithelial Sodium Channel (ENaC) in Cultured Collecting Duct Cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase α (PI5KIα) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIα-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIα decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kα had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIα. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIα enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIα with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIα localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIα.

N-Glycans Mediate Apical Recycling of the Sialomucin Endolyn in Polarized MDCK Cells

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N‐glycan‐dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin‐Darby canine kidney cells. Terminal processing of a subset of endolyns N‐glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine‐based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N‐glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.

Association of gag multimers with filamentous actin during equine infectious anemia virus assembly.

A role for the actin cytoskeleton in retrovirus assembly has long been speculated. However, specific mechanisms by which actin facilitates the assembly process remain elusive. We previously demonstrated differential effects of experimentally modified actin dynamics on virion production of equine infectious anemia virus (EIAV), a lentivirus related to HIV-1, suggesting an involvement of actin dynamics in retrovirus production. In the current study, we used bimolecular fluorescence complementation (BiFC) to reveal intimate (<15 nm) and specific associations between EIAV Gag and actin, but not tubulin. Specific interaction between Gag and filamentous actin was also demonstrated by co-immunoprecipitation experiments combined with the actin severing protein gelsolin to solubilize F-actin. Deletion of capsid (CA) or nucleocapsid (NC) genes reduced Gag association with F-actin by 40% and 95%, respectively. Interestingly, GCN4, a leucine zipper motif, could substitute for the NC domain in mediating F-actin association. Furthermore, deficiency of the DeltaNC Gag in F-actin interaction was restored upon co-expression of Gag constructs containing both CA and NC or the GCN4, suggesting a requirement for Gag polyprotein multimerization prior to F-actin association. The observed Gag-F-actin association appeared to correlate with viral budding, as enhanced budding of the DeltaNC mutant was evident upon restoration of F-actin association. Intracellular association of Gag complexes with F-actin was also detected by immunoscanning electron microscopy of Triton-extracted EIAV-infected cells. Together, these data suggest that Gag multimers induced by CA and NC domains interact with F-actin and that this association is important for efficient virion production.