scholarly journals Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

2009 ◽  
Vol 186 (2) ◽  
pp. 269-282 ◽  
Author(s):  
Glen A. Farr ◽  
Michael Hull ◽  
Ira Mellman ◽  
Michael J. Caplan
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Small Gtpases ◽  
Recycling Endosomes ◽  
Multiple Routes ◽  
Sorting Process ◽  
Polarized Epithelial Cells ◽  
Pass Through ◽  
Golgi Network

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.

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 A dileucine motif in the COOH-terminal domain of NKCC1 targets the cotransporter to the plasma membrane

2019 ◽  
Vol 316 (4) ◽  
pp. C545-C558 ◽  
Author(s):  
Rainelli Koumangoye ◽  
Salma Omer ◽  
Eric Delpire
Keyword(s):  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Terminal Domain ◽  
Late Endosomes ◽  
Dileucine Motif ◽  
Polarized Epithelial Cells ◽  
Epithelial Dysfunction ◽  
Golgi Network

Na+-K+-2Cl− cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl− and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.

scholarly journals Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

2008 ◽  
Vol 19 (5) ◽  
pp. 2059-2068 ◽  
Author(s):  
Lauren Henry ◽  
David R. Sheff
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Recycling Endosome ◽  
Recycling Pathway ◽  
Polarized Epithelial Cells ◽  
Pass Through

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

scholarly journals Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

1998 ◽  
Vol 9 (3) ◽  
pp. 685-699 ◽  
Author(s):  
Kent K. Grindstaff ◽  
Robert L. Bacallao ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Golgi Complex ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Domains ◽  
Trans Golgi Network ◽  
G Cells ◽  
Polarized Epithelial Cells ◽  
Golgi Network

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

scholarly journals Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells

2008 ◽  
Vol 182 (5) ◽  
pp. 845-853 ◽  
Author(s):  
Rita L. Nokes ◽  
Ian C. Fields ◽  
Ruth N. Collins ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Bronchial Epithelial ◽  
Recycling Endosomes ◽  
Cargo Proteins ◽  
Vesicular Stomatitis ◽  
Golgi Network

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.

Polarized biosynthetic traffic in renal epithelial cells: sorting, sorting, everywhere

2006 ◽  
Vol 291 (4) ◽  
pp. F707-F713 ◽  
Author(s):  
Mark A. Ellis ◽  
Beth A. Potter ◽  
Kerry O. Cresawn ◽  
Ora A. Weisz
Keyword(s):  
Epithelial Cells ◽  
Biosynthetic Pathway ◽  
Basolateral Membrane ◽  
Proper Function ◽  
Membrane Domains ◽  
Conventional Model ◽  
Recycling Endosomes ◽  
Sorting Signals ◽  
Vesicular Carriers ◽  
Golgi Network

The maintenance of apical and basolateral membrane domains with distinct protein and lipid compositions is necessary for the proper function of polarized epithelial cells. Delivery of cargo to the basolateral surface is thought to be mediated by the interaction of cytoplasmically disposed sorting signals with sorting receptors, whereas apically destined cargoes are sorted via mechanisms dependent on cytoplasmic, glycan-mediated, or lipid-interacting sorting signals. Apical and basolateral cargo are delivered to the surface in discrete tubular and vesicular carriers that bud from the trans-Golgi network (TGN). While it has long been thought that the TGN is the primary compartment in which apical and basolateral cargoes are segregated, recent studies suggest that sorting may begin earlier along the biosynthetic pathway. Moreover, rather than being delivered directly from the TGN to the cell surface, at least a subset of biosynthetic cargo appears to transit recycling endosomes en route to the plasma membrane. The implications and limitations of these challenges to the conventional model for how proteins are sorted and trafficked along the biosynthetic pathway are discussed.

scholarly journals Myosin VI is required for sorting of AP-1B–dependent cargo to the basolateral domain in polarized MDCK cells

2007 ◽  
Vol 177 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Josephine Sui-Yan Au ◽  
Claudia Puri ◽  
Gudrun Ihrke ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Myosin Vi ◽  
Tail Domain ◽  
Functional Complex ◽  
Polarized Epithelial Cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B–dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.

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