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1.  Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1 
Glycobiology  2013;23(8):935-945.
The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111–1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [35S]Met/Cys or glycans with [3H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [3H]MUC1 when compared with [35S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O-glycan processing from Gal1-4GlcNAc1-6(Gal1-3)GalNAc (core 2) to Gal1-3GalNAc (core 1).
PMCID: PMC3695752  PMID: 23640779
glycan processing; membrane trafficking; MUC1; O-glycosylation; recycling
2.  VAMP7 Modulates Ciliary Biogenesis in Kidney Cells 
PLoS ONE  2014;9(1):e86425.
Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis.
PMCID: PMC3899255  PMID: 24466086
3.  Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization 
Molecular Biology of the Cell  2013;24(12):1996-2007.
The mechanisms that regulate the apical sorting of proteins are unclear, but clustering may play an important role. A role for dimerization and higher-order oligomerization in the biosynthetic transport of the model O-glycosylated protein p75 has been identified. This study also suggests that the O-glycans of p75 have a structural role in apical sorting.
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.
PMCID: PMC3681702  PMID: 23637462
4.  OCRL1 Modulates Cilia Length in Renal Epithelial Cells 
Traffic (Copenhagen, Denmark)  2012;13(9):1295-1305.
Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.
PMCID: PMC3652247  PMID: 22680056
kidney; Lowe syndrome; MDCK; oculocerebrorenal syndrome of Lowe; phosphatidylinositol 4,5-bisphosphate; zebrafish
5.  MUC1 traverses apical recycling endosomes along the biosynthetic pathway in polarized MDCK cells 
Biological chemistry  2009;390(7):551-556.
MUC1 is a heavily glycosylated transmembrane protein localized at the apical surface of polarized epithelial cells. Here, we examined the biosynthetic route of newly synthesized MUC1 in polarized Madin-Darby canine kidney (MDCK) cells. Apically and basolaterally destined cargo are sorted at the trans-Golgi network into distinct vesicles, and proteins with lipid raft-dependent apical targeting signals and glycan-dependent apical targeting signals appear to specifically transit apical early endosomes (AEEs) and apical recycling endosomes (AREs), respectively. Using metabolic labeling we found that MUC1 is efficiently targeted to the apical surface of polarized MDCK cells with a t1/2 of 45 min. Apical delivery was not altered by inactivation of AEEs by treatment with hydrogen peroxide and diaminobenzidine treatment after apical loading of endosomes with horseradish peroxidase-conjugated wheat germ agglutinin. However, expression of a GFP-tagged myosin Vb tail fragment (GFP-MyoVbT) that disrupts export from the ARE significantly reduced MUC1 apical expression. Moreover, MUC1 expressed for brief periods in MDCK cells co-localized with GFP-MyoVbT. We conclude that MUC1 traffics to the apical surface via AREs in polarized renal epithelial cells.
PMCID: PMC3625034  PMID: 19453277
apical; biosynthetic traffic; endosome; glycosylation; Madin-Darby canine kidney (MDCK); MUC1; myosin Vb; polarity
6.  Sorting It Out in Endosomes: An Emerging Concept in Renal Epithelial Cell Transport Regulation 
Physiology (Bethesda, Md.)  2010;25(5):280-292.
Ion and water transport by the kidney is continually adjusted in response to physiological cues. Selective endocytosis and endosomal trafficking of ion transporters are increasingly appreciated as mechanisms to acutely modulate renal function. Here, we discuss emerging paradigms in this new area of investigation.
PMCID: PMC3625043  PMID: 20940433
7.  Multiple Biosynthetic Trafficking Routes for Apically Secreted Proteins in MDCK Cells 
Traffic (Copenhagen, Denmark)  2011;13(3):433-442.
Many newly synthesized membrane proteins traverse endocytic intermediates en route to the surface in polarized epithelial cells; however, the biosynthetic route of secreted proteins has not been elucidated. We monitored the trafficking route of two secreted proteins with different apical sorting signals: the N-glycan dependent cargo glycosylated growth hormone (gGH) and Ensol, a soluble version of endolyn whose apical sorting is independent of N-glycans. Both proteins were observed to colocalize in part with apical recycling endosome (ARE) markers. Cargo that lacks an apical targeting signal and is secreted in a nonpolarized manner did not localize to the ARE. Expression of dominant-negative mutant of Myosin Vb, which disrupts ARE export of glycan dependent membrane proteins, selectively inhibited apical release of gGH but not Ensol. FRAP measurements revealed that gGH in the ARE was less mobile than Ensol, consistent with tethering to a sorting receptor. However, knockdown of galectin-3 or galectin-4, lectins implicated in apical sorting, had no effect on the rate or polarity of gGH secretion. Together, our results suggest that apically-secreted cargo selectively access the ARE and are exported via differentially regulated pathways.
PMCID: PMC3276681  PMID: 22118573
8.  PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells 
PLoS ONE  2013;8(1):e53790.
Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.
PMCID: PMC3547069  PMID: 23342003
9.  Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9–dependent mechanism 
Molecular Biology of the Cell  2012;23(18):3636-3646.
The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Apical delivery of endolyn requires recognition of sialic acids on its N-glycans possibly (or likely) mediated by galectin-9.
The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Along rat kidney tubules, endolyn is variously localized to the apical surface and endosomal/lysosomal compartments. Apical delivery of newly synthesized rat endolyn predominates over direct lysosomal delivery in polarized Madin–Darby canine kidney cells. Apical sorting depends on terminal processing of a subset of lumenal N-glycans. Here we dissect the requirements of N-glycan processing for apical targeting and investigate the underlying mechanism. Modulation of glycan branching and subsequent polylactosamine elongation by knockdown of N-acetylglucosaminyltransferase III or V had no effect on apical delivery of endolyn. In contrast, combined but not individual knockdown of sialyltransferases ST3Gal-III, ST3Gal-IV, and ST6Gal-I, which together are responsible for addition of α2,3- and α2,6-linked sialic acids on N-glycans, dramatically decreased endolyn surface polarity. Endolyn synthesized in the presence of kifunensine, which blocks terminal N-glycan processing, reduced its interaction with several recombinant canine galectins, and knockdown of galectin-9 (but not galectin-3, -4, or -8) selectively disrupted endolyn polarity. Our data suggest that sialylation enables recognition of endolyn by galectin-9 to mediate efficient apical sorting. They raise the intriguing possibility that changes in glycosyltransferase expression patterns and/or galectin-9 distribution may acutely modulate endolyn trafficking in the kidney.
PMCID: PMC3442411  PMID: 22855528
10.  Taking the Scenic Route: Biosynthetic Traffic to the Plasma Membrane in Polarized Epithelial Cells 
Traffic (Copenhagen, Denmark)  2009;10(8):972-981.
The maintenance of epithelial cell function requires the establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct lipid and protein compositions. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans-Golgi network. Recent studies have illuminated additional complexities in the subsequent delivery of these proteins to the cell surface. In particular, multiple routes to the apical and basolateral cell surfaces have been uncovered, and many of these involve indirect passage through endocytic compartments. This review summarizes our current understanding of these routes and discusses open issues that remain to be clarified.
PMCID: PMC2786770  PMID: 19453969
apical; AP-1B; basolateral; biosynthetic delivery; endosome; kidney; lipid raft; MDCK; polarized traffic
11.  HIV-1 Matrix Dependent Membrane Targeting Is Regulated by Gag mRNA Trafficking 
PLoS ONE  2009;4(8):e6551.
Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC) assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.
PMCID: PMC2717210  PMID: 19662089
12.  Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis 
The Journal of Experimental Medicine  2008;205(6):1477-1490.
The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential–mucolipin-1 (TRP-ML1) ion channel. The “biogenesis” model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1–deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1–deficient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV.
PMCID: PMC2413042  PMID: 18504305
13.  Distinct Intracellular Trafficking of Equine Infectious Anemia Virus and Human Immunodeficiency Virus Type 1 Gag during Viral Assembly and Budding Revealed by Bimolecular Fluorescence Complementation Assays▿  
Journal of Virology  2007;81(20):11226-11235.
Retroviral Gag polyproteins are necessary and sufficient for virus budding. Numerous studies of human immunodeficiency virus type 1 (HIV-1) Gag assembly and budding mechanisms have been reported, but relatively little is known about these fundamental pathways among animal lentiviruses. While there may be a general assumption that lentiviruses share common assembly mechanisms, studies of equine infectious anemia virus (EIAV) have indicated alternative cellular pathways and cofactors employed among lentiviruses for assembly and budding. In the current study, we used bimolecular fluorescence complementation to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells. The results of these studies demonstrated that replacing the natural RNA nuclear export element (Rev-response element [RRE]) used by HIV-1 and EIAV with the hepatitis B virus posttranscriptional regulatory element (PRE) altered HIV-1, but not EIAV, Gag assembly sites and budding efficiency in human cells. Consistent with this novel observation, different assembly sites were revealed in human cells for Rev-dependent EIAV and HIV-1 Gag polyproteins. In rodent cells, Rev-dependent HIV-1 Gag assembly and budding were blocked, but changing RRE to PRE rescued HIV-1 Gag assembly and budding. In contrast, EIAV Gag polyproteins synthesized from mRNA exported via either Rev-dependent or PRE-dependent mechanisms were able to assemble and bud efficiently in rodent cells. Taken together, our results suggest that lentivirus assembly and budding are regulated by the RNA nuclear export pathway and that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding.
PMCID: PMC2045577  PMID: 17686839
14.  Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells 
Molecular Biology of the Cell  2007;18(10):3978-3992.
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
PMCID: PMC1995710  PMID: 17686995
15.  Receptor-Mediated Entry by Equine Infectious Anemia Virus Utilizes a pH-Dependent Endocytic Pathway 
Journal of Virology  2005;79(23):14489-14497.
Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.
PMCID: PMC1287590  PMID: 16282448
16.  Specific N-Glycans Direct Apical Delivery of Transmembrane, but Not Soluble or Glycosylphosphatidylinositol-anchored Forms of Endolyn in Madin-Darby Canine Kidney Cells 
Molecular Biology of the Cell  2004;15(3):1407-1416.
The sialomucin endolyn is a transmembrane protein with a unique trafficking pattern in polarized Madin-Darby canine kidney cells. Despite the presence of a cytoplasmic tyrosine motif that, in isolation, is sufficient to mediate basolateral sorting of a reporter protein, endolyn predominantly traverses the apical surface en route to lysosomes. Apical delivery of endolyn is disrupted in tunicamycin-treated cells, implicating a role for N-glycosylation in apical sorting. Site-directed mutagenesis of endolyn's eight N-glycosylation sites was used to identify two N-glycans that seem to be the major determinants for efficient apical sorting of the protein. In addition, apical delivery of endolyn was disrupted when terminal processing of N-glycans was blocked using glycosidase inhibitors. Missorting of endolyn occurred independently of the presence or absence of the basolateral sorting signal, because apical delivery was also inhibited by tunicamycin when the cytoplasmic tyrosine motif was mutated. However, we found that apical secretion of a soluble mutant of endolyn was N-glycan independent, as was delivery of glycosylphosphatidylinositol-anchored endolyn. Thus, specific N-glycans are only essential for the apical sorting of transmembrane endolyn, suggesting fundamental differences in the mechanisms by which soluble, glycosylphosphatidylinositol-anchored, and transmembrane proteins are sorted.
PMCID: PMC363156  PMID: 14699065
17.  Differential Effects of Actin Cytoskeleton Dynamics on Equine Infectious Anemia Virus Particle Production 
Journal of Virology  2004;78(2):882-891.
Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.
PMCID: PMC368807  PMID: 14694119
18.  Basolateral Cycling Mediated by a Lumenal Domain Targeting Determinant 
Molecular Biology of the Cell  2003;14(5):1801-1807.
All identified basolateral sorting signals of integral membrane proteins are cytoplasmically disposed, suggesting that basolateral targeting is mediated exclusively by direct interaction with vesicle coat components. Here, we report that GPP130, a cis-Golgi protein that undergoes endosome-to-Golgi retrieval using the late endosome-bypass pathway in nonpolarized cells, cycles via the basolateral membrane in polarized MDCK cells. Significantly, the membrane-proximal lumenal domain of GPP130, which mediates GPP130 localization and trafficking in nonpolarized cells, was both necessary and sufficient for basolateral cycling in MDCK cells. The use of lumenal determinants for both basolateral cycling and endosome-to-Golgi retrieval suggests that a novel receptor-mediated mechanism operates at both the trans-Golgi network and distal sites to sort GPP130 along the late-endosome-bypass retrieval pathway in polarized cells.
PMCID: PMC165078  PMID: 12802056
19.  Influenza M2 Proton Channel Activity Selectively Inhibits Trans-Golgi Network Release of Apical Membrane and Secreted Proteins in Polarized Madin-Darby Canine Kidney Cells 
The Journal of Cell Biology  2000;148(3):495-504.
The function of acidification in protein sorting along the biosynthetic pathway has been difficult to elucidate, in part because reagents used to alter organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in protein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We expressed the influenza virus M2 protein, an acid-activated ion channel that equilibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral proteins. M2 activity affects the pH of only a subset of acidified organelles, and its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G. Apodaca, Y. Altschuler, S. Hardy, and O.A. Weisz. 1998. Mol. Biol. Cell. 8:2477–2490; Henkel, J.R., J.L. Popovich, G.A. Gibson, S.C. Watkins, and O.A. Weisz. 1999. J. Biol. Chem. 274:9854–9860). M2 expression significantly decreased the kinetics of cell surface delivery of the apical membrane protein influenza hemagglutinin, but not of the basolaterally delivered polymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of γ-glutamyltranspeptidase were reduced with no effect on the basolaterally secreted fraction. Interestingly, M2 activity had no effect on the rate of secretion of a nonglycosylated protein (human growth hormone [hGH]) that was secreted equally from both surfaces. However, M2 slowed apical secretion of a glycosylated mutant of hGH that was secreted predominantly apically. Our results suggest a role for acidic trans-Golgi network pH in signal-mediated loading of apical cargo into forming vesicles.
PMCID: PMC2174804  PMID: 10662775
acidification; polarity; Madin-Darby; canine kidney; influenza M2; apical
20.  Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells 
Molecular Biology of the Cell  1998;9(9):2477-2490.
The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.
PMCID: PMC25516  PMID: 9725907
21.  Clathrin-mediated Endocytosis of MUC1 Is Modulated by Its Glycosylation State 
Molecular Biology of the Cell  2000;11(3):819-831.
MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.
PMCID: PMC14813  PMID: 10712502
22.  Efficient Export of the Vesicular Stomatitis Virus G Protein from the Endoplasmic Reticulum Requires a Signal in the Cytoplasmic Tail That Includes Both Tyrosine-based and Di-acidic Motifs 
Molecular Biology of the Cell  2000;11(1):13-22.
The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxØ (where x is any amino acid and Ø is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.
PMCID: PMC14753  PMID: 10637287

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