Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

Interactions between the exocytic and endocytic pathways in polarized Madin-Darby canine kidney cells.

The compartments involved in polarized exocytosis of membrane proteins are not well defined. In this study we hypothesized that newly synthesized polymeric immunoglobulin receptors are targeted from the trans-Golgi network to endosomes prior to their appearance on the basolateral cell surface of polarized Madin-Darby canine kidney cells. To examine this hypothesis, we have used an assay designed to measure the meeting of newly synthesized receptors with a selective population of apical or basolateral endosomes loaded with horseradish peroxidase. We found that in the course of basolateral exocytosis, the wild-type polymeric immunoglobulin receptor is targeted from the trans-Golgi network to apical and basolateral endosomes. Phosphorylation of a Ser residue in the cytoplasmic tail of the receptor is implicated in this process. The biosynthetic pathway of apically sorted polymeric immunoglobulin receptor mutants similarly traversed apical endosomes, raising the possibility that apical receptors are segregated from basolateral receptors in apical endosomes. The post-endocytic pathway of transcytosing and recycling receptors also passed through apical endosomes. Together, these observations are consistent with the possibility that the biosynthetic and endocytic routes merge into endosomes and justify a model suggesting that endosomal recycling processes govern polarized trafficking of proteins traveling in both pathways.

Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling.

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medical recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome.

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.

Interactions of the AP-1 Golgi adaptor with the polymeric immunoglobulin receptor and their possible role in mediating brefeldin A-sensitive basolateral targeting from the trans-Golgi network.

We provide morphological, biochemical, and functional evidence suggesting that the AP-1 clathrin adaptor complex of the trans-Golgi network interacts with the polymeric immunoglobulin receptor in transfected Madin-Darby canine kidney cells. Our results indicate that immunofluorescently labeled gamma-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. gamma-Adaptin is co-immunoisolated with membranes expressing the wild-type receptor. The entire AP-1 adaptor complex could be chemically cross-linked to the receptor in filter-grown cells. gamma-Adaptin could be co-immunoprecipitated with the wild-type receptor, with reduced efficiency with receptor mutant whose basolateral sorting motif has been deleted, and not with receptor lacking its cytoplasmic tail. Co-immunoprecipitation of gamma-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with AP-1 but did not abrogate the fidelity of its basolateral targeting from the trans-Golgi network. However, the kinetics of receptor delivery to the basolateral cell surface were slowed by the mutation. Although surface delivery of the wild-type receptor was inhibited by brefeldin A, the delivery of the mutant receptor was insensitive to the drug. Our results are consistent with a working model in which phosphorylated cytoplasmic serine modulates the recruitment of the polymeric immunoglobulin receptor into AP-1/clathrin-coated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from the trans-Golgi network.

Glycosylation of a vesicular monoamine transporter: a mutation in a conserved proline residue affects the activity, glycosylation, and localization of the transporter.

The role of N-glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [3H]serotonin. Part of this effect was due to a general toxic effect of the drug. Therefore, to assess the contribution of each of the glycosylation sites to the transporter activity, the three putative N-glycosylation sites were mutated individually, in combination, and in toto ("triple" mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, which retained at least 50% of the wild-type activity. No significant differences were found either in the time dependence of uptake or the apparent affinity for ligands of the triple mutant compared with the wild-type protein. It is interesting that in contrast to plasma-membrane neurotransmitter transporters, the unglycosylated form of rVMAT1 distributed in the cell as the wild-type protein. Pro43 is a highly conserved residue located at the beginning of the large loop in which all the potential glycosylation sites are found. A Pro43Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylation sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro43Leu mutant clearly differed from that of the wild type. In contrast, a Pro43Gly mutant displayed an activity practically identical to the wild-type protein. As this replacement generated a protein with wild-type characteristics, we suggest that the conformation conferred by the amino acid at this position is essential for activity.

The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains.

Basolateral sorting of the polymeric immunoglobulin receptor (pIgR) expressed in Madin-Darby canine kidney (MDCK) cells is mediated by a 17-residue sorting signal that resides in the cytoplasmic domain. We have recently analyzed the sequence requirements of the signal by alanine scanning mutagenesis. We found that basolateral sorting is mediated primarily by three amino acids: H656, R657 and V660. Individual mutations of each of these residues to Ala caused a substantial decrease in basolateral sorting and a corresponding increase in targeting to the apical surface. Structural analysis of 17-residue peptides corresponding to the signal revealed that V660 is in a beta-turn (probably type I) secondary structure, and its mutation to Ala destabilized the turn. H656 and R657 were not part of the turn and substitution of Arg657 to Ala had no effect on the turn stability. These results suggested that the signal is comprised of two structurally distinct domains: a critical V660 in the context of the beta-turn and an additional two residues (H656 and R657) that are not in the turn and probably are unimportant for its stability. Here we provide evidence suggesting that the two domains are distinguishable not only by their structure but also by their function. Basolateral targeting of pIgR mutants bearing Ala mutations at either 656 or 657 was not affected by treatment with brefeldin A (BFA), while basolateral targeting of pIgR containing an Ala substitution at position 660 was markedly and uniquely stimulated by BFA. Compared to single Ala substitutions, simultaneous mutations of H656 and R657 to Ala caused an additional minor effect on basolateral and apical sorting, whereas double mutations of V660 and either H656 or R657 resulted in a maximal decrease in basolateral targeting and corresponding increase in apical targeting. These results suggest the existence of two domains in the signal. When both domains are destroyed, basolateral targeting is maximally inhibited. The results also imply that V660 mediates basolateral sorting by a different mechanism from H656 and R657. We suggest that V660 and perhaps more generally the beta-turn may interact with BFA-sensitive adaptor complexes.

Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor.

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca(2+)-dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is the most prominent 35S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.

Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids.

Polarized epithelial cells can sort plasma membrane proteins to the apical or basolateral domain either by direct targeting from the trans-Golgi network (TGN) or by targeting to one surface, followed by endocytosis and transcytosis to the opposite surface. In Madin-Darby canine kidney (MDCK) cells, targeting of the polymeric immunoglobulin receptor (pIgR) to the basolateral surface is controlled by a sorting signal residing in the membrane proximal 17 amino acids of the cytoplasmic domain of this receptor. We have recently found that individual mutations at any of three residues in this signal, His656, Arg657 and Val660, substantially decrease targeting from the TGN to the basolateral surface and correspondingly increase targeting from the TGN to the apical surface. Here we report that these mutations decrease the recycling of basolaterally endocytosed pIgR to that surface, and correspondingly increase its transcytosis to the apical surface. This effect occurred in mutant pIgRs that either contained the full-length cytoplasmic domain or were truncated to contain only the 17-residue basolateral targeting signal, and was independent of phosphorylation of pIgR at Ser664. Our results indicate that polarized sorting of the pIgR in the endocytotic and exocytotic pathways are controlled by the same amino acids.

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide.

Brefeldin-A inhibits the delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells.

We have studied the effects of brefeldin A (BFA) on the polarized delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells. Unlike the delivery of several other basolateral membrane and secretory proteins, the delivery of the polymeric immunoglobulin receptor from the trans-Golgi network to the cell surface was inhibited by BFA. The effect of BFA treatment was apparent at 1.0 microgram/ml (36% inhibition), and maximal inhibition was observed at 10 micrograms/ml (70% inhibition). The delivery of the receptor from the endoplasmic reticulum to the basolateral surface was even more sensitive to the effect of BFA; delivery was inhibited 95% in cells treated with 1 micrograms/ml BFA. The selective action of BFA on the basolateral delivery of the polymeric immunoglobulin receptor suggests that there may be multiple pathways for delivery of proteins to the basolateral cell surface of MDCK cells.

Transient alterations in the lateral mobility of erythrocyte membrane components during Sendai virus-mediated fusion.

Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.

Accumulation of Sendai virus glycoproteins in cell-cell contact regions and its role in cell fusion.

Lateral motion of the viral envelope proteins in the target cell membrane was shown recently to be essential for cell fusion by Sendai virus (Henis, Y. I., Herman-Barhom, Y., Aroeti, B., and Gutman, O. (1989) J. Biol. Chem. 264, 17119-17125). To explore the mechanism that gives rise to this requirement, we have now investigated the distribution of Sendai virus envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) on human erythrocytes in the course of fusion, using fluorescence microscopy and image analysis. In these studies, both the F and the HN proteins were found to accumulate in cell-cell contact regions, on the time scale of the fusion process. We propose that migration of the viral glycoproteins to cell contact regions and accumulation at the contact sites are essential parts of the fusion mechanism and form the basis to the requirement for their lateral motion in the fusion event.

Rotational mobility of Sendai virus glycoproteins in membranes of fused human erythrocytes and in the envelopes of cell-bound virions.

The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (ER)-labeled monovalent Fab' antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab'-viral glycoprotein complexes was studied both in the envelopes of unfused virions bound to erythrocyte ghosts and in the target cell membrane after fusion had occurred. The rotational correlation times (phi) of Er-Fab'-labeled F and HN were rather similar in the envelopes of bound unfused virions, but highly different in membranes of fused cells. The different phi values indicate that F and HN diffuse separately in the target cell membrane and for the major part are not complexed together. The temperature dependence of the phi values of the Er-Fab'-viral glycoprotein complexes revealed a breakpoint at 22 degrees C for the F protein both in bound virions and in the membranes of fused cells, and for the HN proteins in the envelopes of bound virions. In all these cases, the phi values increased between 4 and 22 degrees C, demonstrating a reduction in the rate of rotational diffusion. Further elevation of the temperature reversed the direction of the change in phi. This phenomenon may reflect a temperature-dependent microaggregation of F and HN saturating at ca. 22 degrees C and presumably related to the fusion mechanism since the breakpoint temperature correlates closely with the threshold temperature for virus-cell and cell-cell fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Lateral mobility of both envelope proteins (F and HN) of Sendai virus in the cell membrane is essential for cell-cell fusion.

Fluorescence photobleaching recovery was employed to study the effects of specific immobilization of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin-neuraminidase) on the virally mediated fusion of human erythrocytes. Lateral immobilization of varying fractions of F and/or HN (after virus adsorption and hemagglutination, but before fusion) was achieved by cross-linking them with succinyl concanavalin A (inhibiting both F and HN) or with specific rabbit IgG directed against either F or HN. Alternatively, agglutinated cells were treated with low concentrations of the above proteins (inducing only minor inhibition of either mobility or fusion), and immobilization of F and/or HN was induced by cross-linking with a secondary antibody; this protocol ensured a minimal contribution of direct binding to the viral proteins to the inhibition of fusion. Our results demonstrate that lateral immobilization of either F or HN results in a strong inhibition of cell-cell fusion and a much weaker inhibition of virus-cell fusion. The level of cell-cell fusion was directly correlated with the level of laterally mobile viral glycoproteins in the cell membrane (either F or HN). We conclude that lateral mobility of both F and HN in the red cell membrane is essential for cell-cell fusion and that not only F but also HN has a role in this fusion event. The possible reasons for the different dependence of cell-cell and virus-cell fusion on viral glycoprotein mobility are discussed.

Effects of fusion temperature on the lateral mobility of Sendai virus glycoproteins in erythrocyte membranes and on cell fusion indicate that glycoprotein mobilization is required for cell fusion.

In order to investigate the requirement for lateral mobilization of viral envelope glycoproteins on the cell surface in the induction of cell-cell fusion, we employed fluorescence photobleaching recovery to study the effect of the fusion temperature on the lateral mobilization of Sendai virus glycoproteins in the human erythrocyte membrane. As the fusion temperature was reduced below 37 degrees C (to 31 or 25 degrees C), the rates of virus-cell fusion, the accompanying hemolysis, and cell-cell fusion were all slowed down. However, the plateau (final level) after the completion of fusion was significantly reduced at lower fusion temperatures only in the case of cell-cell fusion, despite the rather similar final levels of virus-cell fusion. A concomitant decrease as a function of the fusion temperature was observed in the fraction of cell-associated viral glycoproteins that became laterally mobile in the erythrocyte membrane during fusion, and a strict correlation was found between the level of laterally mobile viral glycoproteins in the cell membrane and the final extent of cell-cell fusion. The accompanying reduction in the lateral diffusion coefficients (D) of the viral glycoproteins (1.4-fold at 31 degrees C and 1.9-fold at 25 degrees C, as compared to 37 degrees C) does not appear to determine the final level of cell-cell fusion, since fusing the cells with a higher amount of virions at 25 degrees C increased the final level of cell-cell fusion while D remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Fusion of native Sendai virions with human erythrocytes. Quantitation by fluorescence photobleaching recovery.

We have recently developed a method to quantitate the fusion of reconstituted viral envelopes with cells by fluorescence photobleaching recovery (FPR) (Aroeti, B & Henis, Y I, Biochemistry 25 (1986) 4588). The method is based on the incorporation of non quenching concentrations of the fluorescent lipid probe N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)phosphatidylethanolamine during the reconstitution of the viral envelopes (the latter probe does not incorporate efficiently into the membrane of native virions). In the present work, we employed the fluorescent dye octadecyl rhodamine B chloride (R18), which can be incorporated directly into the membrane of native enveloped virions, to extend the FPR method to study fusion between native Sendai virions and intact human erythrocytes. The R18 fluorescence was found to be quenched in the viral envelope at the concentration range required for the FPR experiments, possibly due to preferential insertion of the probe into specific domains in the viral membrane. We therefore developed a correction (presented in the Appendix) which takes into account the lower quantum yield of the probe molecules in the membranes of unfused virions in the calculation of the fraction of fused virions from the FPR experiments. The results demonstrate that the method does indeed measure virus-cell fusion, and that the contribution of exchange to the measurements is not significant. The applicability of the method was further verified by the similarity of the results to those obtained independently by fluorescence dequenching measurements, and its ability to measure the distribution of virus-cell fusion within the cell population was demonstrated. These results suggest that the use of R18 can enlarge the scope of the FPR experiments to study the fusion of native virions with cells.