Cellular uptake of amelogenin, and its localization to CD63, and Lamp1-positive vesicles.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11).

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

Vesicular trafficking machinery, the actin cytoskeleton, and H+-K+-ATPase recycling in the gastric parietal cell.

Gastric HCl secretion by the parietal cell involves the secretagogue-regulated re-cycling of the H+-K+-ATPase at the apical membrane. The trafficking of the H+-K+-ATPase and the remodelling of the apical membrane during this process are likely to involve the co-ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H+-K+-ATPase and of components of the actin-based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein- protein interactions that regulate apical membrane trafficking in many other epithelial cells.

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

Clathrin in mitotic spindles.

Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit.

The assembly of the beta-subunit of the gastric H-K-ATPase (HKbeta) with the alpha-subunit of the H-K-ATPase or the Na-K-ATPase (NaKalpha) was characterized with two anti-HKbeta monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKbeta, MAb 2/2E6 was observed to bind to HKbeta only when interactions between alpha- and beta-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S(226)LHY(229) of the extracellular domain of HKbeta. The epitope for MAb 2G11 was mapped to the eight NH(2)-terminal amino acids of the cytoplasmic domain of HKbeta. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKbeta with alpha-subunits of the endogenous cell surface NaKalpha, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKbeta. In HKbeta-transfected LLC-PK(1) cells, significant immunofluorescent labeling of HKbeta at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKbeta could also be coimmunoprecipitated with NaKalpha. Thus assembly of HKbeta with NaKalpha does not appear to be a stringent requirement for cell surface delivery of HKbeta in LLC-PK(1) cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid alpha-beta heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKbeta that is associated with alpha-beta interaction.

MHC class II molecules, cathepsins, and La/SSB proteins in lacrimal acinar cell endomembranes.

Sjögren's syndrome is a chronic autoimmune disease affecting the lacrimal glands and other epithelia. It has been suggested that acinar cells of the lacrimal glands provoke local autoimmune responses, leading to Sjögren's syndrome when they begin expressing major histocompatibility complex (MHC) class II molecules. We used isopycnic centrifugation and phase partitioning to resolve compartments that participate in traffic between the basolateral membranes and the endomembrane system to test the hypothesis that MHC class II molecules enter compartments that contain potential autoantigens, i.e., La/SSB, and enzymes capable of proteolytically processing autoantigen, i.e., cathepsins B and D. A series of compartments identified as secretory vesicle membranes, prelysosomes, and microdomains of the trans-Golgi network involved in traffic to the basolateral membrane, to the secretory vesicles, and to the prelysosomes were all prominent loci of MHC class II molecules, La/SSB, and cathepsins B and D. These observations support the thesis that lacrimal gland acinar cells that have been induced to express MHC class II molecules function as autoantigen processing and presenting cells.

Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1.

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

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.

Stimulation with carbachol alters endomembrane distribution and plasma membrane expression of intracellular proteins in lacrimal acinar cells.

The events that lead to Sjögren's autoimmune processes in the lacrimal gland remain poorly understood. The acinar cell's responses to acute cholinergic stimulation include release of secretory products across the apical plasma membrane (apm) and a number of processes related to traffic between endomembrane compartments and the basal-lateral plasma membranes (blm), such as recruitment of Na, K-ATPase, accelerated recycling, and accelerated transcytosis of secretory IgA. We tested the hypothesis that stimulation-induced acceleration of endomembrane traffic is accompanied by changes in compartmentation and increased blm expression of proteins that are normally sequestered in endomembrane compartments. Isolated rabbit lacrimal gland acinar cells were cultured in serum-free media for 2 days. After harvesting, cells were incubated with or without 10 microm carbachol at 37 degrees C for 20 min. Cells were lysed, and lysates were analysed by isopycnic centrifugation on sorbitol gradients. Galactosyltransferase catalytic activity was determined biochemically. Different forms of cathepsin B were detected by Western blotting. Carbachol stimulation decreased the contents of beta-hexosaminidase, alpha-glucosidase, and protein in secretory vesicles and increased them in specific compartments of the trans-Golgi network (ld-tgns). Stimulation also caused levels of galactosyltransferase, preprocathepsin B, and procathepsin B to increase two- to three-fold in the blm as well as increasing in the ld-tgns. Other changes caused by sustained stimulation included: (a) increased levels of protein and procathepsin B in compartments of the lysosomal pathway; (b) changes in the distributions of Rab5 within the endomembrane system; (c) changes in the distribution of Rab6 within the Golgi complex and tgn; (d) decreased expression of acid phosphatase and MHC class II molecules in the blm; and (e) decreased total content of Na,K-ATPase, which appeared to have been selectively depleted from the tgn and blmre. We propose that the normal compartmentation of certain proteins may allow them to remain cryptic, such that they are not subject to central tolerance. Stimulation-induced increases in the levels expressed at the blm or secreted to the interstitium may, therefore, contribute to initiation of local autoimmune responses.

An immunologically distinct beta-adaptin on tubulovesicles of gastric oxyntic cells.

Clathrin and the gamma-adaptin subunit of the AP-1 clathrin adaptor have been previously identified on H-K-ATPase-rich tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 (Cell Physiol. 43): C1017-C1029]. We further characterized this AP-1 adaptor from rabbit and hog tubulovesicles biochemically and immunologically. Clathrin coat proteins were stripped from purified tubulovesicular membranes and fractionated by hydroxyapatite chromatography. The AP-1 adaptor appears to elute at 200 mM sodium phosphate, based on the presence of proteins in this fraction that are immunoreactive with antibodies against three of the four subunits of this heterotetrameric complex: the gamma-, mu1-, and sigma1-adaptin subunits. Although the putative beta-adaptin subunit in this fraction is not immunoreactive with the anti-beta-adaptin monoclonal antibody (MAb), this beta-adaptin is immunoreactive with polyclonal antibodies (PAbs) directed against the peptide sequence Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro- Val640 , a region conserved between beta1- and beta2-adaptins that is thought to be involved in the binding of clathrin heavy chain. Immunoprecipitation of the AP-1 adaptor complex from this fraction with anti-gamma-adaptin MAb 100/3 resulted in the coimmunoprecipitation of the beta-adaptin that did not react with the anti-beta-adaptin MAb but did react with the anti-beta-adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a beta-adaptin that was recognized by both the anti-beta-adaptin MAb and PAbs. These results suggest that the tubulovesicular AP-1 adaptor complex may be distinct from that found in the trans-Golgi network and may contain an immunologically distinct beta-adaptin. This immunologically distinct beta-adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1.

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells.

gamma-Adaptin and clathrin heavy chain were identified on tubulovesicles of gastric oxyntic cells with the anti-gamma-adaptin monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb (MAb 23), respectively. In Western blots, crude gastric microsomes from rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for gamma-adaptin and clathrin. In immunofluorescent labeling of isolated rabbit gastric glands, anti-gamma-adaptin and anti-clathrin heavy chain immunoreactivity appeared to be concentrated in oxyntic cells. In primary cultures of rabbit oxyntic cells, the immunocytochemical distribution of gamma-adaptin immunoreactivity was similar to that of the tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase. Further biochemical characterization of the tubulovesicular gamma-adaptin-containing complex suggested that it has a subunit composition that is typical of that for a clathrin adaptor: in addition to the gamma-adaptin subunit, it contains a beta-adaptin subunit and other subunits of apparent molecular masses of 50 kDa and 19 kDa. From solubilized gastric microsomes from rabbit, gamma-adaptin could be copurified with the major cargo protein of tubulovesicles, the H-K-ATPase. Thus this tubulovesicular coat may bind directly to the H-K-ATPase and may thereby mediate the regulated trafficking of the H-K-ATPase at the apical membrane of the oxyntic cell during the gastric acid secretory cycle. Given the similarities of the regulated trafficking of the H-K-ATPase with recycling of cargo through the apical recycling endosome of many epithelial cells, we propose that tubulovesicular clathrin and adaptors may regulate some part of an apical recycling pathway in other epithelial cells.

Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface.

CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane-endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjögren's syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjögren's autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses.

Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a case of mistaken identity.

Na-K-ATPase is associated with a variety of membrane populations in lacrimal acinar cells. Acinus-like structures formed by rabbit acinar cells in primary culture were incubated with horseradish peroxidase (HRP) to label basolateral and endosomal membranes and then analyzed by electron microscopy cytochemistry with the 3-3'-diaminobenzidine reaction or by fractionation and measurement of marker catalytic activities or immunoreactivities. HRP adsorbed to basolateral membranes at 4 degrees C. Fractionation showed it associated with low-density membranes enriched in acid phosphatase and TGN38 but containing only minor amounts of Na-K-ATPase. Cells internalized HRP to cytoplasmic vesicles, Golgi structures, and lysosomes at 37 degrees C. The major endosomal compartment revealed by fractionation coincided with major peaks of Na-K-ATPase and Rab6 and secondary peaks of galactosyltransferase and gamma-adaptin. Carbachol (10 microM) increased lysosomal and Golgi labeling. Thus most of the Na-K-ATPase is located in the basolateral membrane-oriented endosomal system, concentrated in a compartment possibly related to the trans-Golgi network. Constitutive and stimulation-accelerated traffic to and from this compartment may serve several exocrine cell functions.

Rapid internalization of the polymeric immunoglobulin receptor requires phosphorylated serine 726.

S726 of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) resides within a consensus sequence for phosphorylation by protein kinases A, G, and C, and casein kinase II. Mutation of S726 to Ala and expression of this mutant pIgR in Madin-Darby canine kidney cells results in a receptor in which steady-state phosphorylation is reduced to 49% of wild-type levels. This mutant receptor is also significantly impaired in its internalization from the basolateral membrane. During the first minute, internalization of radioiodinated ligands (either dIgA or monovalent anti-pIgR Fabs) by this mutant pIgR is only 35% of that by wild-type pIgR. Internalization of unoccupied mutant receptors is similarly inhibited. Delivery of newly made mutant receptor from the trans-Golgi network to the basolateral surface is completely normal. The only other trafficking step inhibited by this mutation is the transcytosis of radioiodinated dIgA. Within 2 h, the mutant pIgR will transcytose 58% of a preinternalized cohort of dIgA, while the wild-type transcytoses 76%. This inhibition of transcytosis may be an indirect consequence of impaired internalization. The correlation between the loss of phosphorylation and inhibition of internalization suggests that phosphorylation of S726 may represent a novel mechanism for regulation of internalization of the pIgR.