PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.     

Isolation of exocytic carrier vesicles from BHK cells

Newly synthesized cell surface glycoproteins are transported from the trans-Golgi network (TGN) to the plasma membrane in vesicular carriers. Here we describe a cell-free system in which the formation of these carrier vesicles is reconstituted. Vesicle formation and release occurred specifically from the TGN and were dependent on ATP and cytosol. The released vesicles were isolated by density gradient sedimentation and specific immunoadsorption. Electron microscopy demonstrated that the vesicles had a diameter of 84 +/- 6 nm. The immunoisolated vesicles had a highly simplified protein pattern on two-dimensional gel electrophoresis.     

Structural basis of human erythrocyte glucose transporter function in reconstituted vesicles

The transmembrane orientation of the human erythrocyte glucose transporter was assessed based on polarized Fourier transform infrared and ultraviolet circular dichroism spectroscopic data obtained from oriented multilamellar films of the reconstituted transporter vesicles. Infrared spectra revealed that there are distinct vibrations for alpha-helical structure while the vibrational frequencies specific to beta-structure are characteristically absent. Analysis of linear dichroism of the infrared spectra further indicated that these alpha-helices in the transporter are preferentially oriented perpendicular to the lipid bilayer plane forming an effective tilt of less than 38 degrees from the membrane normal. Such a preferential orientation was further supported by ultraviolet circular dichroism spectra which reveal that the 208 nm Moffit band found in the detergent-solubilized preparation is absent in the film preparation. Linear dichroism data further indicated that D-glucose, a typical substrate, further reduces this effective tilt angle slightly.     

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.     

Membrane fusion correlates with surface charge in exocytic vesicles

Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion.     

Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo

The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

Release of putative exocytic transport vesicles from perforated MDCK cells.

Mechanically perforated MDCK cells were used to study membrane transport between the trans-Golgi network and the apical and basolateral plasma membrane domains in vitro. Three membrane transport markers--an apical protein (fowl plague virus haemagglutinin), a basolateral protein (vesicular stomatitis virus G protein), and a lipid marker destined for both domains (C6-NBD-sphingomyelin)--were each accumulated in the trans-Golgi by a 20 degrees C block of transport and their behaviour monitored following cell perforation and incubation at 37 degrees C. In the presence of ATP and in the absence of calcium ions a considerable fraction of the transport markers were released from the perforated cells in sealed membrane vesicles. Control experiments showed that the vesicles were not generated by non-specific vesiculation of the Golgi complex or the plasma membrane. The vesicles had well defined sedimentation properties and the orientation expected of transport vesicles derived from the trans-Golgi network.     

Role of insulin-dependent cortical fodrin/spectrin remodeling in glucose transporter 4 translocation in rat adipocytes

Fodrin or nonerythroid spectrin is an abundant component of the cortical cytoskeletal network in rat adipocytes. Fodrin has a highly punctate distribution in resting cells, and insulin causes a dramatic remodeling of fodrin to a more diffuse pattern. Insulin-mediated remodeling of actin occurs to a lesser extent than does that of fodrin. We show that fodrin interacts with the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 4, and this interaction is increased by insulin stimulation and decreased by prior latrunculin A treatment. Latrunculin A disrupts all actin filaments, inhibits glucose transporter 4 (GLUT4) translocation, and causes fodrin to partially redistribute from the plasma membrane to the cytosol. In contrast, cytochalasin D disrupts only the short actin filament signal, and cytochalasin D neither inhibits GLUT4 translocation nor fodrin redistribution in adipocytes. Together, our data suggest that insulin induces remodeling of the fodrin-actin network, which is required for the fusion of GLUT4 storage vesicles with the plasma membrane by permitting their access to the t-SNARE syntaxin 4.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.     

Phosphatidylinositol 4-kinase type IIalpha is targeted specifically to cellugyrin-positive glucose transporter 4 vesicles

Phosphoinositides now emerge as important regulators of membrane traffic. In particular, phosphatidylinositol 4-phosphate may serve as a precursor for polyphosphorylated derivatives of phosphatidylinositol and, also, may regulate vesicular traffic by recruiting specific proteins to the membrane. Early results have demonstrated the presence of phosphatidylinositol 4-kinase (PI4K) activity in glucose transporter 4 (Glut4) vesicles from fat and skeletal muscle cells. However, the molecular identity of phosphatidylinositol 4-kinase(s) associated with Glut4 vesicles has not been characterized. It has also been determined that Glut4 vesicles are not homogeneous and represent a mixture of at least two vesicular populations: ubiquitous cellugyrin-positive transport vesicles and specialized cellugyrin-negative insulin-responsive Glut4 storage vesicles, which are different in size, protein composition, and functional properties. Using sequential immunoadsorption, subcellular fractionation, and immunofluorescence staining, we show that virtually all PI4K activity in Glut4 vesicles is represented by PI4K type IIalpha, which is associated with cellugyrin-positive vesicles and is not detectable in the Glut4 storage vesicles. The unique N terminus of PI4K type IIalpha is required for the targeting of the enzyme to cellugyrin-positive vesicles. Knockdown of PI4K type IIalpha with the help of short hairpin RNA does not decrease the amount of cellugyrin-positive vesicles in human embryonic kidney 293 cells.     

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.     

Functional asymmetry of the human Na+/glucose transporter (hSGLT1) in bacterial membrane vesicles

The functional characteristics of the forward and reverse transport modes of the human Na(+)/glucose transporter (hSGLT1) were investigated using plasma membrane vesicles of E. coli expressing the recombinant transporter. Correctly and inverse-oriented vesicles were employed to measure the initial rates of methyl-alpha-D-glucose uptake, under zero-trans conditions, as a function of Na(+), sugar, and phlorizin concentrations and membrane potential. This approach enabled the analysis of the two faces of hSGLT1 in parallel, revealing the reversibility of Na(+)/sugar cotransport. While the key characteristics of secondary active sugar transport were maintained in both modes, namely, Na(+) and voltage dependence, the kinetic properties of the two sides indicated a functional asymmetry of the transporter. That is, the apparent affinity for sugar and driver cation Na(+) exhibited a difference of more than 1 order of magnitude between the two modes. Furthermore, the selectivity pattern of ligands and the interaction of the transporter with the competitive inhibitor phlorizin were different. Whereas the high-affinity substrates, D-glucose and D-galactose, inhibited uptake of radioactive sugar tracer at their physiological concentrations (10 mM) in the forward reaction, they were poor inhibitors even at high concentrations in the reverse transport mode. Taken together, these results confirm the successful employment of E. coli to express and characterize a human membrane protein (hSGLT1), elucidating the functional asymmetry of this cotransporter.     

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.     

Phosphatidylinositol 4-kinase is a component of glucose transporter (GLUT 4)-containing vesicles.

We recently developed a procedure for immunoisolating insulin-responsive membrane vesicles that contain the muscle/fat glucose transporter isoform, GLUT 4, from rat adipocytes. Utilizing this methodology, we are analyzing the components of these vesicles to gain an understanding of how they are regulated by insulin. In this report we identify a phosphatidylinositol (PtdIns) 4-kinase as a constituent of glucose transporter vesicles (GTVs). This kinase has the biochemical and immunological properties of a type II PtdIns 4-kinase as classified by Endeman et al. (Endemann, G., Dunn, S. N., and Cantley, L. C. (1987) Biochemistry 26, 6845-6852). A monoclonal antibody, 4C5G, which specifically inhibits the type II PtdIns 4-kinase, suppresses 80% of the GTV-PtdIns 4-kinase activity. In addition, the GTVs-PtdIns 4-kinase is maximally activated by the nonionic detergent Triton X-100, at a concentration of 0.2% and is inhibited by adenosine with a Ki of approximately 20-30 microM. We find that the GTVs do not contain any PtdIns4P 5-kinase or diacylglycerol kinase activities, whereas these activities were detected in the plasma membrane. An analysis of the subcellular distribution of PtdIns 4-kinase activity in the rat adipocyte shows that there are similar levels of activity in GTVs, plasma membranes, and the high and low density microsomal fractions, whereas the mitochondria- and nuclei-containing fractions have less than 5% of the activity seen in other fractions. Low density microsomes were subfractionated by sucrose density gradient centrifugation and PtdIns 4-kinase activity was found to correlate closely with the distribution of membrane protein, indicating that the activity is equally distributed throughout this heterogenous population of membranes. PtdIns 4-kinase activity measured in GTVs, plasma membranes, and low density microsomes, was not affected by prior treatment of the intact adipocytes with 35 nM insulin. We postulate that while the GTV-PtdIns 4-kinase is not regulated by insulin, it may play a role in defining the fusogenic properties necessary to mediate membrane movement between the GTVs, plasma membranes, and microsomes.     

Pigment containing lipid vesicles

Summary It was shown previously (Walz, 1976) that chlorophylla incorporated into the membrane of lecithin vesicles is a probe which detects the aggregational state of the lipids. This phenomenon is interpreted in terms of a solvatochromism, i.e., the effect of various solvents on the absorption spectrum of the solute. The sensor characteristics can be expressed by a set of solvatochromic coefficients, which are pertinent to the electronic transitions occuring in chlorophylla on excitation with light, and by means of the absorption bands associated with these transitions. An unambiguous resolution of spectra into absorption bands is not yet practicable, but at least part of the bands can be approximated by, Gaussian components which then allows us to estimate the solvatochromic coefficients From these data and based on the currently available theoretical and experimental information about, solvatochromism, it is concluded that the chromophore, i.e., the porphyrin ring of chlorophylla, is located adjacent to the glycerol-ester moieties of the lecithin molecules in the membrane, and that the sensor ability relies on different orientations of the chromophore for lecithin in different states of aggregation.     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Localization of the GLUT1 glucose transporter to brefeldin A-sensitive vesicles of differentiated CIT3 mouse mammary epithelial cells

Glucose is a precursor of lactose, the major carbohydrate and osmotic constituent of human milk, which is synthesized in the Golgi. The GLUT1 glucose transporter is the only glucose transporter isoform expressed in the mammary gland. The hypothesis that lactogenic hormones induce GLUT1 and cause its localization to the Golgi of mammary epithelial cells was tested in CIT(3)mouse mammary epithelial cells. Treatment with prolactin and hydrocortisone caused a 15-fold induction of GLUT1 by Western blotting, but 2-deoxyglucose uptake decreased. Subcellular fractionation and density gradient centrifugation demonstrated enrichment of Golgi fractions with GLUT1. Lactogenic hormones enhanced GLUT1 glycosylation, but did not determine whether GLUT1 was targeted to plasma membrane or to Golgi. Confocal microscopy revealed that lactogenic hormones alter GLUT1 targeting from a plasma membrane pattern to a predominant perinuclear distribution with punctate scattering through the cytoplasm. GLUT1 is targeted to a compartment which is more sensitive to Brefeldin A than the compartments in which GM130 and beta-COP reside. Targeting of GLUT1 to endosomes was specifically excluded. We conclude that prolactin and hydrocortisone induce GLUT1, enhance GLUT1 glycosylation, and cause glycosylation-independent targeting of GLUT1 to Brefeldin A-sensitive vesicles which may represent a subcompartment of cis-Golgi. These results demonstrate a hormonally-regulated targeting mechanism for GLUT1 and are consistent with an important role for GLUT1 in the provision of substrate for lactose synthesis.