scholarly journals Basolateral Cycling Mediated by a Lumenal Domain Targeting Determinant

2003 ◽  
Vol 14 (5) ◽  
pp. 1801-1807 ◽  
Author(s):  
Manojkumar A. Puthenveedu ◽  
Jennifer R. Bruns ◽  
Ora A. Weisz ◽  
Adam D. Linstedt
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Direct Interaction ◽  
Late Endosome ◽  
Sorting Signals ◽  
Golgi Protein ◽  
Necessary And Sufficient ◽  
Golgi Network ◽  
Lumenal Domain ◽  
Basolateral Targeting

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.

scholarly journals Basolateral Targeting of ERBB2 Is Dependent on a Novel Bipartite Juxtamembrane Sorting Signal but Independent of the C-Terminal ERBIN-Binding Domain

2002 ◽  
Vol 22 (18) ◽  
pp. 6553-6563 ◽  
Author(s):  
Christian Dillon ◽  
Anna Creer ◽  
Karen Kerr ◽  
Angelika Kümin ◽  
Clive Dickson
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Binding Domain ◽  
Neurotrophin Receptor ◽  
P75 Neurotrophin Receptor ◽  
Endosomal Trafficking ◽  
Membrane Domain ◽  
Sorting Signal ◽  
Necessary And Sufficient ◽  
Basolateral Targeting

ABSTRACT ERBB2 is a receptor tyrosine kinase present on the basolateral membrane of polarized epithelia and has important functions in organ development and tumorigenesis. Using mutagenic analyses and Madin-Darby canine kidney (MDCK) cells, we have investigated the signals that regulate basolateral targeting of ERBB2. We show that basolateral delivery of ERBB2 is dependent on a novel bipartite juxtamembrane sorting signal residing between Gln-692 and Thr-701. The signal shows only limited sequence homology to known basolateral targeting signals and is both necessary and sufficient for correct sorting of ERBB2. In addition we demonstrate that this motif can function as a dominant basolateral targeting signal by its ability to redirect the apically localized P75 neurotrophin receptor to the basolateral membrane domain of polarized epithelial cells. Interestingly, LLC-PK1 cells, which are deficient for the μ1B subunit of the AP1B adaptor complex, missort a large proportion of ERBB2 to the apical membrane domain. This missorting can be partially corrected by the introduction of μ1B, suggesting a possible role for AP1B in ERBB2 endosomal trafficking. Furthermore, we find that the C-terminal ERBIN binding domain of ERBB2 is not necessary for its basolateral targeting in MDCK cells.

scholarly journals GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

2006 ◽  
Vol 172 (7) ◽  
pp. 1023-1034 ◽  
Author(s):  
Simona Paladino ◽  
Thomas Pocard ◽  
Maria Agata Catino ◽  
Chiara Zurzolo
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Biochemical Assays ◽  
Intracellular Sorting ◽  
Apical Sorting ◽  
Golgi Network ◽  
Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

scholarly journals Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

1992 ◽  
Vol 118 (1) ◽  
pp. 51-62 ◽  
Author(s):  
S H Low ◽  
B L Tang ◽  
S H Wong ◽  
W Hong
Keyword(s):  
Protein Targeting ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Selective Inhibition ◽  
Surface Expression ◽  
Apical Surface ◽  
Golgi Transport ◽  
Apical Domain ◽  
Golgi Network ◽  
Basolateral Targeting

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

scholarly journals A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 471-479 ◽  
Author(s):  
M J Rindler ◽  
M G Traber
Keyword(s):  
Epithelial Cells ◽  
Secretory Pathway ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Plasma Membrane ◽  
Intestinal Epithelial ◽  
Polarized Secretion ◽  
Sorting Signals

Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

scholarly journals Ubc9 interacts with Lu/BCAM adhesion glycoproteins and regulates their stability at the membrane of polarized MDCK cells

2007 ◽  
Vol 402 (2) ◽  
pp. 311-319 ◽  
Author(s):  
Emmanuel Collec ◽  
Wassim El nemer ◽  
Emilie Gauthier ◽  
Pierre Gane ◽  
Marie-Christine Lecomte ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Ex Vivo ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Direct Interaction ◽  
Cytoplasmic Tail ◽  
Protein Accumulation ◽  
Membrane Expression

Lu (Lutheran) blood group and BCAM (basal cell adhesion molecule) antigens both reside on two gp (glycoprotein) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in RBCs (red blood cells), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST (glutathione S-transferase) pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK (Madin–Darby canine kidney) epithelial cells respectively. Replacement of Lys585 by alanine in the Lu gp abolished in vitro and ex vivo interactions of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared with wild-type Lu. Pulse–chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our results support a physiological role for the direct interaction between Lu gp and Ubc9 protein and reveal a role for this enzyme in regulating the stability of Lu gp at the cell membrane.

Lin-7 targets the Kir 2.3 channel on the basolateral membrane via a L27 domain interaction with CASK

2007 ◽  
Vol 293 (6) ◽  
pp. C1733-C1741 ◽  
Author(s):  
Christine Alewine ◽  
Bo-young Kim ◽  
Vandana Hegde ◽  
Paul A. Welling
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Pdz Domain ◽  
Endocytic Pathway ◽  
Membrane Expression ◽  
Interacting Protein ◽  
Pdz Proteins ◽  
Calcium Calmodulin Dependent ◽  
Anchoring Protein ◽  
Basolateral Targeting

Polarized expression of the Kir 2.3 channel in renal epithelial cells is influenced by the opposing activities of two different PDZ proteins. Mammalian Lin-7 (mLin-7) directly interacts with Kir 2.3 to coordinate basolateral membrane expression, whereas the tax interacting protein 1 (TIP-1), composed of a single PDZ domain, competes for interaction with mLin-7 and drives Kir 2.3 into the endocytic pathway. Here we show that the basolateral targeting function of mLin-7 depends on its L27 domain, which directs interaction with a cognate L27 domain in the basolateral membrane-anchoring protein, calcium/calmodulin-dependent serine protein kinase (CASK). In MDCK cells, the expression of an mLin-7 mutant that lacks the L27 domain displaced Kir 2.3 from the mLin-7/CASK complex and caused the channel to accumulate into large intracellular vesicles that partially colocalized with Rab-11. Conversely, transplantation of the mLin-7 L27 domain to TIP-1 conferred CASK interaction and basolateral targeting of Kir 2.3. Expression of the CASK L27 domain redistributed endogenous mLin-7 to an intracellular compartment and caused Kir 2.3 to accumulate in subapical endosomes. Taken together, these data support a model whereby mLin-7 acts as a PDZ-to-L27 adapter, mediating indirect association of Kir 2.3 with a basolateral membrane scaffold and thereby stabilizing Kir 2.3 at the basolateral membrane.

scholarly journals The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

1998 ◽  
Vol 142 (5) ◽  
pp. 1245-1256 ◽  
Author(s):  
Jen-Zen Chuang ◽  
Ching-Hwa Sung
Keyword(s):  
Secretory Pathway ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Apical Plasma Membrane ◽  
Glutathione S Transferase ◽  
Sorting Signals ◽  
Apical Sorting

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

Constitutive secretion of a bacterial enzyme by polarized epithelial cells

1992 ◽  
Vol 102 (3) ◽  
pp. 495-504 ◽  
Author(s):  
K.L. Soole ◽  
J. Hall ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Quantitative Detection ◽  
Bacterial Protein ◽  
Endoglucanase Activity ◽  
Sorting Signals ◽  
Bacterial Enzyme ◽  
Constitutive Secretion

The constitutive (or default) pathway for protein secretion was investigated in two epithelial cells, Madin-Darby canine kidney (MDCK) and human colonic adenocarcinoma (Caco-2), using a bacterial enzyme. The choice of a bacterial protein was based on the requirement to identify a protein devoid of sorting signals. The sorting of a bacterial endoglucanase derived from Clostridium thermocellum, endoglucanase E, from stably transfected MDCK and Caco-2 cells was examined. The choice of a bacterial endoglucanase for these studies has advantages of simple, sensitive and quantitative detection, while higher eukaryotic cells do not express endoglucanase activity. Both cell lines secreted a 50 kDa form of the bacterial protein, while smaller intracellular forms were also observed. In polarized layers of MDCK cells the endoglucanase was secreted into both membrane domains in the ratio 62% apical and 38% basolateral. In Caco-2 cells secretion was predominantly, 70%, through the basolateral membrane. These results define the constitutive pathway for protein secretion in these two model epithelial cells.

scholarly journals Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells.

1995 ◽  
Vol 128 (3) ◽  
pp. 321-332 ◽  
Author(s):  
S Höning ◽  
W Hunziker
Keyword(s):  
Mdck Cells ◽  
Transmembrane Protein ◽  
Cytoplasmic Tail ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Lysosomal Sorting ◽  
Lysosomal System ◽  
Golgi Network ◽  
Basolateral Targeting ◽  
Cytoplasmic Determinants

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.

scholarly journals Aminopeptidase N is directly sorted to the apical domain in MDCK cells.

1990 ◽  
Vol 111 (6) ◽  
pp. 2923-2930 ◽  
Author(s):  
H P Wessels ◽  
G H Hansen ◽  
C Fuhrer ◽  
A T Look ◽  
H Sjöström ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Cell Types ◽  
Aminopeptidase N ◽  
Apical Surface ◽  
Apical Domain ◽  
Single Protein ◽  
Cell Type Specific ◽  
Golgi Network

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from the trans-Golgi-network to the apical membrane has been demonstrated. However, different proteins had been studied in these cells. To compare the sorting of a single protein in both systems, we have expressed aminopeptidase N, which already had been shown to be sorted indirectly in hepatocytes, in transfected MDCK cells. As expected, it was predominantly localized to the apical domain of the plasma membrane. By monitoring the appearance of newly synthesized aminopeptidase N at the apical and basolateral surface, it was found to be directly sorted to the apical domain in MDCK cells, indicating that the sorting pathways are indeed cell type-specific.

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