scholarly journals Arf6 regulates AP-1B–dependent sorting in polarized epithelial cells

2011 ◽  
Vol 194 (6) ◽  
pp. 873-887 ◽  
Author(s):  
Elina Shteyn ◽  
Lucy Pigati ◽  
Heike Fölsch
Keyword(s):  
Epithelial Cells ◽  
Dominant Negative ◽  
Recycling Endosomes ◽  
Membrane Recruitment ◽  
Important Regulator ◽  
Basolateral Plasma Membrane ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Basolateral Targeting

The epithelial cell–specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B’s clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B–dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B’s functions in polarized epithelial cells.

scholarly journals Differential Recognition of Tyrosine-based Basolateral Signals by AP-1B Subunit μ1B in Polarized Epithelial Cells

2002 ◽  
Vol 13 (7) ◽  
pp. 2374-2382 ◽  
Author(s):  
Hisashi Sugimoto ◽  
Masayuki Sugahara ◽  
Heike Fölsch ◽  
Yasuhiro Koide ◽  
Fubito Nakatsu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Mutant Form ◽  
Targeting Signals ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Basolateral Targeting

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit μ1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of μ1B. The mutant (M-μ1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit μ2 are critical for interacting with tyrosine-based endocytosis signals. We show M-μ1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-μ1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on μ1B or M-μ1B expression. Our results suggest that μ1B interacts with different classes of basolateral targeting signals in distinct ways.

scholarly journals Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Ian C. Fields ◽  
Shelby M. King ◽  
Elina Shteyn ◽  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Amino Acid Residues ◽  
Recycling Endosomes ◽  
C Terminus ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

scholarly journals ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells

2011 ◽  
Vol 193 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Epithelial Cells ◽  
Autosomal Recessive ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipoprotein Receptors ◽  
Recycling Endosomes ◽  
Sorting Motif ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Autosomal Recessive Hypercholesterolemia

The autosomal recessive hypercholesterolemia protein (ARH) is well known for its role in clathrin-mediated endocytosis of low-density lipoprotein receptors (LDLRs). During uptake, ARH directly binds to the FxNPxY signal in the cytoplasmic tail of LDLR. Interestingly, the same FxNPxY motif is used in basolateral exocytosis of LDLR from recycling endosomes (REs), which is facilitated by the epithelial-specific clathrin adaptor AP-1B. However, AP-1B directly interacts with neither the FxNPxY motif nor the second more distally located YxxØ sorting motif of LDLR. Here, we show that ARH colocalizes and cooperates with AP-1B in REs. Knockdown of ARH in polarized epithelial cells leads to specific apical missorting of truncated LDLR, which encodes only the FxNPxY motif (LDLR-CT27). Moreover, a mutation in ARH designed to disrupt the interaction of ARH with AP-1B specifically abrogates exocytosis of LDLR-CT27. We conclude that in addition to its role in endocytosis, ARH cooperates with AP-1B in basolateral exocytosis of LDLR from REs.

scholarly journals A Novel Clathrin Adaptor Complex Mediates Basolateral Targeting in Polarized Epithelial Cells

Cell ◽  
1999 ◽  
Vol 99 (2) ◽  
pp. 189-198 ◽  
Author(s):  
Heike Fölsch ◽  
Hiroshi Ohno ◽  
Juan S Bonifacino ◽  
Ira Mellman
Keyword(s):  
Epithelial Cells ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Basolateral Targeting

scholarly journals Novel Membrane Protein shrew-1 Targets to Cadherin-Mediated Junctions in Polarized Epithelial Cells

2004 ◽  
Vol 15 (1) ◽  
pp. 397-406 ◽  
Author(s):  
Sanita Bharti ◽  
Heike Handrow-Metzmacher ◽  
Silvia Zickenheiner ◽  
Andreas Zeitvogel ◽  
Rudolf Baumann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Protein ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Potential Candidate ◽  
Putative Open Reading Frame ◽  
Polarized Epithelial Cells ◽  
E Cadherin

While searching for potential candidate molecules relevant for the pathogenesis of endometriosis, we discovered a 2910-base pair cDNA encoding a novel putative 411-amino acid integral membrane protein that we called shrew-1. The putative open-reading frame was confirmed with antibodies against shrew-1 peptides that labeled a protein of ∼48 kDa in extracts of shrew-1 mRNA-positive tissue and also detected ectopically expressed shrew-1. Expression of epitope-tagged shrew-1 in epithelial cells and analysis by surface biotinylation and immunoblots demonstrated that shrew-1 is indeed a transmembrane protein. Shrew-1 is able to target to E-cadherin-mediated adherens junctions and interact with the E-cadherin–catenin complex in polarized MCF7 and Madin-Darby canine kidney cells, but not with the N-cadherin–catenin complex in nonpolarized epithelial cells. Direct interaction of shrew-1 with β-catenin in in vitro pull-down assay suggests that β-catenin might be one of the proteins that targets and/or retains shrew-1 in the adherens junctions. Interestingly, shrew-1 was partially translocated in response to scatter factor (ligand of receptor tyrosine kinase c-met) from the plasma membrane to the cytoplasm where it still colocalized with endogenous E-cadherin. In summary, we introduce shrew-1 as a novel component of adherens junctions, interacting with E-cadherin–β-catenin complexes in polarized epithelial cells.

scholarly journals A PDZ-Binding Motif Controls Basolateral Targeting of Syndecan-1 Along the Biosynthetic Pathway in Polarized Epithelial Cells

Traffic ◽  
2008 ◽  
Vol 9 (11) ◽  
pp. 1915-1924 ◽  
Author(s):  
Sandra Maday ◽  
Eric Anderson ◽  
Henry C. Chang ◽  
James Shorter ◽  
Ayano Satoh ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Biosynthetic Pathway ◽  
Binding Motif ◽  
Polarized Epithelial Cells ◽  
Basolateral Targeting

scholarly journals M1 Protein Triggers a Phosphoinositide Cascade for Group A Streptococcus Invasion of Epithelial Cells

2003 ◽  
Vol 71 (10) ◽  
pp. 5823-5830 ◽  
Author(s):  
Sai Sudha Purushothaman ◽  
Beinan Wang ◽  
P. Patrick Cleary
Keyword(s):  
Epithelial Cells ◽  
Group A Streptococcus ◽  
Dominant Negative ◽  
M Protein ◽  
Lipid Kinase ◽  
Cytoskeletal Rearrangement ◽  
M1 Protein ◽  
Serovar Typhimurium ◽  
Group A

ABSTRACT Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics. M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A Streptococcus (GAS) and also renders the bacterium resistant to phagocytosis. In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry. LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by Salmonella enterica serovar Typhimurium. Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS. To separate the influence of other streptococcal virulence factors from M protein, Lactococcus lactis was engineered to express M1 protein on its surface. L. lactis(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion. Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells. LY294002 and wortmannin inhibited these cellular changes. A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS. Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.

scholarly journals Regulation of Membrane Trafficking by a Novel Cdc42-related Protein in Caenorhabditis elegans Epithelial Cells

2005 ◽  
Vol 16 (4) ◽  
pp. 1629-1639 ◽  
Author(s):  
S. Jenna ◽  
M.-E. Caruso ◽  
A. Emadali ◽  
D. T. Nguyên ◽  
M. Dominguez ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Rho Gtpases ◽  
Related Protein ◽  
Recycling Endosomes ◽  
C Elegans ◽  
Apical Trafficking ◽  
Golgi Network

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C6-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.

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