Apical Sorting Research Articles

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells.

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station.

The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells

A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5' nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface.

Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

Thematic Review Series: Glycosylphosphatidylinositol (GPI) Anchors: Biochemistry and Cell Biology Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling

Glycosylphosphatidylinositols (GPIs) act as membrane anchors of many eukaryotic cell surface proteins. GPIs in various organisms have a common backbone consisting of ethanolamine phosphate (EtNP), three mannoses (Mans), one non-N-acetylated glucosamine, and inositol phospholipid, whose structure is EtNP-6Manα-2Manα-6Manα-4GlNα-6myoinositol-P-lipid. The lipid part is either phosphatidylinositol of diacyl or 1-alkyl-2-acyl form, or inositol phosphoceramide. GPIs are attached to proteins via an amide bond between the C-terminal carboxyl group and an amino group of EtNP. Fatty chains of inositol phospholipids are inserted into the outer leaflet of the plasma membrane. More than 150 different human proteins are GPI anchored, whose functions include enzymes, adhesion molecules, receptors, protease inhibitors, transcytotic transporters, and complement regulators. GPI modification imparts proteins with unique characteristics, such as association with membrane microdomains or rafts, transient homodimerization, release from the membrane by cleavage in the GPI moiety, and apical sorting in polarized cells. GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertilization, and immune system. Mutations in genes involved in remodeling of the GPI lipid moiety cause human diseases characterized by neurological abnormalities. Yeast Saccharomyces cerevisiae has >60 GPI-anchored proteins (GPI-APs). GPI is essential for growth of yeast. In this review, we discuss biosynthesis of GPI-APs in mammalian cells and yeast with emphasis on the lipid moiety.

ID: 61: Apical and basolateral sorting of IL-11R facilitates co-sorting of gp130

Interleukin (IL-)6 and IL-11 signal via homodimeric gp130 receptors, IL-6R α and IL-11R α , respectively. Preferential basolateral localization was demonstrated for the IL-6R and gp130. IL-11R is expressed in polarized cells cells as well. Moreover, IL-11 is involved in development of gastric tumors and regeneration of colon and heart tissue. Here, we show that the heterologous expression of IL-11R1 resulted in apical and basolateral membrane localization in Madin-Darby canine kidney (MDCK) cells. Transfer of the intracellular domain of the IL-6R to the IL-11R1 results in sorting of the chimeric protein to the basolateral membrane. After stimulation with IL-6 phosphorylation of signal transducer and activator of transcription-3 (STAT-3) was induced via basolaterally located IL-6R-gp130 complexes but not from the apical site. In the absence of IL-6R, gp130-induced STAT3-phosphorylation was also only induced after basolateral application of Hyper-IL-6, a fusion protein of IL-6 and the soluble IL-6R. STAT3 activation of IL-11 was, however, induced from apical and basolateral site. Exchange of the intracellular domains of IL-6R and IL-11R1, renders chimeric IL-11R only responsive from the basolatal site and chimeric IL-6R responsive from the apical and basolateral site. After expression of IL-11R1, MDCK cells became responsive to Hyper-IL-6 from the apical site, suggest that IL-11R1 expression resulted in re-sorting of gp130 also to the apical site. Finally, our data demonstrate that both receptors IL-6R and IL-11R1 are internalized with comparable kinetics by clathrin-mediated endocytosis. Our data showed, that IL-11R is expressed on apical and basolateral membranes which results in abrogation of basolateral sorting of gp130.

Recycling of galectin-3 in epithelial cells

Galectins, a family of β-galactoside binding proteins, do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. They use a so-called unconventional secretion mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. The β-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium. The lectin re-enters the cell by non-clathrin mediated endocytosis and passages through endosomal organelles. This internalized galectin-3 plays an important role in apical protein trafficking by directing the subcellular targeting of apical glycoproteins via oligomerization into high molecular weight clusters, a process that can be fine-tuned by changes in the environmental pH. Following release at the apical plasma membrane, the lectin can reenter the cell for another round of recycling and apical protein sorting. This review will briefly address galectin-3-functions in epithelia and focus on distinct phases in apical recycling of the lectin.

Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubularorgans.

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.

Abstract 5351: The carcinogenesis rate is increased in MAL knock-out mice

Abstract Background: The MAL gene was originally identified by Alonso during a study for differentially expressed genes in T-cell development. The MAL protein, which spans the membrane four times, is involved in normal apical transport and accurate sorting in distinct epithelial cells. The recent study on MAL gene down-regulation in primary human epithelial malignancies suggest that the loss of the MAL gene may be closely linked with a variety of human epithelial malignancies. In our previous study, we revealed that MAL was remarkably down-regulated in human head and neck squamous cell carcinoma. Exogenous expression of MAL in HNSCC cell lines could inhibit the proliferation, invasion, and induce apoptosis of cancer cells in vitro and suppress tumor growth in vivo. These data support that MAL, as a candidate tumor suppressor, contributes to carcinogenesis and development of HNSCC. Methods: To further confirm the role of MAL gene in development of carcinoma, a MAL gene knockout mouse model was produced. And the genotype and phenotype was investigated by our study group. The genotypes of MAL-/- and MAL+/+ mice were identified by RT-PCR, Western blot and Immunohistochemistry. The histopathology of main organs and tissues (esophagus, stomach, small intestine, colon, oral mucosa, etc.) were analyzed with H & E staining. Chemical carcinogenesis agent (4-nitroquinoline-N-oxide, 4NQO) was used to induce oral mucosa carcinomas. Results: MAL gene knockout mouse model was successfully established and passed on from generation to generation more than 3 years. The genotypes of MAL-/-, MAL+/-, MAL+/+ mice were identified. Phenotypic investigation found that MAL-/- mice raised no obvious spontaneous phenotype in 12 months and the incidence of spontaneous tumor was zero. Nevertheless, we found that there were significant differences between MAL-/- and MAL+/+ mice in the incidence rate, occurrence time and growth speed of tongue and esophageal squamous carcinoma which induced by a low dose of 4NQO water feeding for 16 weeks (P<0.01). We also found that the occurrence of esophageal squamous cell carcinoma performed the same changes as oral mucosa. Conclusions: Our results suggest that MAL gene knockout mouse significantly increased the susceptibility of cancer. The molecular mechanism about the tumor susceptibility becomes more important, and will be significant for developing cancer prevention and treatment methods. Citation Format: Wantao Chen, Xiangbing Wu, Wei Cao, Xu Wang. The carcinogenesis rate is increased in MAL knock-out mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5351. doi:10.1158/1538-7445.AM2014-5351

Influenza A virus hemagglutinin and neuraminidase mutually accelerate their apical targeting through clustering of lipid rafts.

In polarized epithelial cells, influenza A virus hemagglutinin (HA) and neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical plasma membrane for viral assembly and budding. Previous studies have indicated that the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA are required for association with lipid rafts, but the raft dependencies of their apical targeting are controversial. Here, we show that coexpression of HA with NA accelerated their apical targeting through accumulation in lipid rafts. HA was targeted to the apical plasma membrane even when expressed alone, but the kinetics was much slower than that of HA in infected cells. Coexpression experiments revealed that apical targeting of HA and NA was accelerated by their coexpression. The apical targeting of HA was also accelerated by coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and the deletion of the CT in HA and NA that reduced their association with lipid rafts abolished the acceleration of their apical transport, indicating that the lipid raft association is essential for efficient apical trafficking of HA and NA. An in situ proximity ligation assay (PLA) revealed that HA and NA were accumulated and clustered in the cytoplasmic compartments only when both were associated with lipid rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings. We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism of the accelerated apical transport of HA and NA via clustering of lipid rafts. Lipid rafts serve as sites for viral entry, particle assembly, and budding, leading to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase (NA) of influenza virus, key players for particle assembly, contain determinants for apical sorting and lipid raft association. However, it remains to be elucidated how lipid rafts contribute to the apical trafficking and budding. We investigated the relation of lipid raft association of HA and NA to the efficiency of apical trafficking. We show that coexpression of HA and NA induces their accumulation in lipid rafts and accelerates their apical targeting, and we suggest that the accelerated apical transport likely occurs by clustering of lipid rafts at the TGN. This finding provides the first evidence that two different raft-associated viral proteins induce lipid raft clustering, thereby accelerating apical trafficking of the viral proteins.

Clathrin and its adaptor AP1 are required for apical sorting of GPI‐anchored proteins in biosynthetic and recycling routes in MDCK cells (598.4)

To fulfill their functions, epithelial cells establish asymmetric protein distributions in apical and basolateral plasma membranes. To date, the role of clathrin and its adaptor, the AP1 complex, has been documented exclusively for basolateral protein trafficking in mammalian epithelial cells. We found that the apical localization of endogenous glycosylphosphatidylinositol (GPI)‐anchored proteins is severely affected by gene silencing of the clathrin heavy chain and components of the AP1 adaptor complex in MDCK cells. In these cells, apical targeting is affected not only for GPI‐ anchored proteins but also for endogenous secretory proteins and the apical‐destined t‐SNARE, syntaxin 3. Analysis of apical secretion of lysoGPI‐APs reveals that the biosynthetic delivery of GPI‐APs toward apical plasma membrane is impaired in these cells. Furthermore, apical recycling and transcytosis from basolateral to apical membranes of GPI‐APs are also impaired in these cells. Our results formally demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.Grant Funding Source: Swiss National Sciences Foundation Assistant Professorship

Elucidating a mechanism for maltooligosacchride sensing at the small intestine enterocyte (1039.9)

As the primary products of starch digestion by pancreatic α‐amylase, maltose and maltooligosaccharides, are the dominant substrates for α‐glucosidases at the intestinal brush border. Here, immunobloting and biosynthetic labeling using [35S]‐methionine revealed maltose induces the formation of a higher molecular weight (HMW) sucrose‐isomaltase (SI) species with a higher rate for early trafficking and maturation which is more associated with Triton X‐100 lipid rafts, resulting in higher apical sorting of these species. As this finding is potentially indicative of a maltose sensing ability by enterocytes, our goal is to investigate the sensing mechanism on the small intestine enterocytes for maltose sensing leading to the processing of HMW SI. Mass spectrometry study and endoglycosidase treatment of HMW SI showed the increased molecular weight of SI is a consequence of altered N‐ and O‐glycosylation in the Golgi apparatus causing higher apical sorting of SI. In addition, metabolomics study on metabolites involved in differentiation along with transepithelial electric resistance and endoplasmic reticulum stress measurements reveled cells maintain their differentiation through an adaptation to utilize maltose as an energy source. Thus, maltose sensing ability of enterocytes influences the intracellular processing of SI, speculatively to enhance their digestive property.

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.

Galectin-4-mediated transcytosis of transferrin receptor.

Some native epithelia, for example, retinal pigment epithelium (RPE) and kidney proximal tubule (KPT), constitutively lack the basolateral sorting adaptor AP-1B; this results in many basolateral plasma membrane proteins being repositioned to the apical domain, where they perform essential functions for their host organs. We recently reported the underlying apical polarity reversal mechanism: in the absence of AP-1B-mediated basolateral sorting, basolateral proteins are shuttled to the apical plasma membrane through a transcytotic pathway mediated by the plus-end kinesin KIF16B. Here, we demonstrate that this apical transcytotic pathway requires apical sorting of basolateral proteins, which is mediated by apical signals and galectin-4. Using RPE and KPT cell lines, and AP-1B-knockdown MDCK cells, we show that mutation of the N-glycan linked to N727 in the basolateral marker transferrin receptor (TfR) or knockdown of galectin-4 inhibits TfR transcytosis to apical recycling endosomes and the apical plasma membrane, and promotes TfR lysosomal targeting and subsequent degradation. Our results report a new role of galectins in basolateral to apical epithelial transcytosis.

PH‐Dependent Recycling of Galectin‐3 at the Apical Membrane of Epithelial Cells

The β-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium by non-classical secretion. Once within the apical extracellular milieu, galectin-3 can re-enter the cell followed by passage through endosomal organelles and modulate apical protein sorting. Here, we could show that galectin-3 is internalized by non-clathrin mediated endocytosis. Within endosomal organelles this pool associates with newly synthesized neurotrophin receptor in the biosynthetic pathway and assists in its membrane targeting. This recycling process is accompanied by transient interaction of galectin-3 with detergent insoluble membrane microdomains in a lactose- and pH-dependent manner. Moreover, in the presence of lactose, apical sorting of the neurotrophin receptor is affected following endosomal deacidification. Taken together, our results suggest that internalized galectin-3 directs the subcellular targeting of apical glycoproteins by membrane recycling.

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75-green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.

Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin-Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

Maturation and trafficking of a HMW sucrase‐isomaltase species expressed via maltose sensing

As the primary product of starch digestion by pancreatic α‐amylase, maltose and maltooligosaccharides, are the dominant substrates for α‐glucosidases at the intestinal brush border. Here, immunoblotting of SI from Caco‐2 cells showed maltose induce the formation of a higher molecular weight (HMW) SI species. As this finding is potentially indicative of a maltose sensing ability by enterocytes, our goal is to investigate the molecular mechanisms associated with the maturation and trafficking of this HMW SI. Biosynthetic labeling using [35S]‐methionine revealed a higher rate for early trafficking and maturation of the HMW SI species. Endoglycosidase treatment of the immunoprecipitated SI showed the increased molecular weight of SI is a consequence of altered N‐ and O‐glycosylation of this enzyme. In comparison to the control, the HMW SI was found to be more associated with Triton X‐100 lipid rafts which results in higher apical sorting of these species, as defined by isolation and characterization of the brush border membrane of Caco‐2 cells. Thus, maltose sensing ability of enterocytes influences the intracellular processing of the glycoproteins, speculatively to enhance their digestive property.

The apical sorting signal for human GLUT9b resides in the N-terminus

The two splice variants of human glucose transporter 9 (hGLUT9) are targeted to different polarized membranes. hGLUT9a traffics to the basolateral membrane, whereas hGLUT9b traffics to the apical region. This study examines the sorting mechanism of these variants, which differ only in their N-terminal domain. Mutating a di-leucine motif unique to GLUT9a did not affect targeting. Chimeric proteins were made using GLUT1, a basolaterally targeted transporter, and GLUT3, an apically targeted protein whose signal lies in the C-terminus. Overexpression of the chimeric proteins in polarized cells demonstrates that the N-terminus of hGLUT9b contains a signal capable of redirecting GLUT1 to the apical membrane. The N-terminus of hGLUT9a, however, does not contain a basolateral signal sufficient enough to redirect GLUT3. Portions of the GLUT9a N-terminus were substituted with corresponding portions of the GLUT9b N-terminus to determine the motif responsible for apical targeting. The first 16 amino acids were not found to be a sufficient apical signal. The last ten amino acids of the N-termini differ only in amino-acid class at one location. In the B-form, leucine, a hydrophobic residue, is substituted for lysine, a basic residue, found in the A-form. However, mutation of the leucine in hGLUT9b to a lysine resulted in retention of the apical signal. We therefore believe the apical signal exists as an interplay between the final ten amino acids of the N-terminus and another motif within the protein such as the intracellular loop or other motifs within the N-terminus.

Oligomerization is required for normal endocytosis/transcytosis of a GPI-anchored protein in polarized hepatic cells

Targeting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) in polarized epithelial cells depends on their association with detergent-resistant membrane microdomains called rafts. In MDCK cells, GPI-APs associate with rafts in the trans-Golgi network and are directly delivered to the apical membrane. It has been shown that oligomerization is required for their stabilization in rafts and their apical targeting. In hepatocytes, GPI-APs are first delivered to the basolateral membrane and secondarily reach the apical membrane by transcytosis. We investigated whether oligomerization is required for raft association and apical sorting of GPI-APs in polarized HepG2 cells, and at which step of the pathway oligomerization occurs. Model proteins were wild-type GFP-GPI and a double cysteine GFP-GPI mutant, in which GFP dimerization was impaired. Unlike wild-type GFP-GPI, which was efficiently endocytosed and transcytosed to the apical surface, the double cysteine mutant was basolaterally internalized, but massively accumulated in early endosomes, and reached the bile canaliculi with delayed kinetics. The double cysteine mutant was less resistant to Triton X-100 extraction, and formed fewer high molecular weight complexes. We conclude from these results that, in hepatocytes, oligomerization plays a key role in targeting GPI-APs to the apical membrane, by increasing their affinity for rafts and allowing their transcytosis.