scholarly journals The Receptor Recycling Pathway Contains Two Distinct Populations of Early Endosomes with Different Sorting Functions

1999 ◽  
Vol 145 (1) ◽  
pp. 123-139 ◽  
Author(s):  
David R. Sheff ◽  
Elizabeth A. Daro ◽  
Michael Hull ◽  
Ira Mellman
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Receptor Recycling ◽  
Membrane Domain ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Basolateral Plasma Membrane ◽  
Recycling Pathway ◽  
Plasma Membrane Domain ◽  
Two Populations

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.

scholarly journals Transferrin receptor recycling in the absence of perinuclear recycling endosomes

2002 ◽  
Vol 156 (5) ◽  
pp. 797-804 ◽  
Author(s):  
David Sheff ◽  
Laurence Pelletier ◽  
Christopher B. O'Connell ◽  
Graham Warren ◽  
Ira Mellman
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Mammalian Cells ◽  
Receptor Recycling ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Essential Components ◽  
Recycling Pathway ◽  
Dynamic Structures ◽  
Peripheral Cytoplasm

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

scholarly journals Interaction with mLin-7 Alters the Targeting of Endocytosed Transmembrane Proteins in Mammalian Epithelial Cells

2001 ◽  
Vol 12 (5) ◽  
pp. 1329-1340 ◽  
Author(s):  
Samuel W. Straight ◽  
Liguang Chen ◽  
David Karnak ◽  
Ben Margolis
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Targeting ◽  
Mdck Cells ◽  
Pdz Domain ◽  
Receptor Protein ◽  
Membrane Domain ◽  
Basolateral Plasma Membrane ◽  
Carboxyl Terminal ◽  
Plasma Membrane Domain

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein–protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.

scholarly journals Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells

1997 ◽  
Vol 137 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Sven C.D. van IJzendoorn ◽  
Mirjam M.P. Zegers ◽  
Jan Willem Kok ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Hepg2 Cells ◽  
Accurate Estimation ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Dibutyryl Camp ◽  
Preferential Localization ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Fluorescent Lipid

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chainlabeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37°C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi–related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

The PDZ-interaction of the intestinal anion exchanger downregulated in adenoma (DRA; SLC26A3) facilitates its movement into Rab11a-positive recycling endosomes

2013 ◽  
Vol 304 (11) ◽  
pp. G980-G990 ◽  
Author(s):  
S. Lissner ◽  
C.-J. Hsieh ◽  
L. Nold ◽  
K. Bannert ◽  
P. Bodammer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Dominant Negative Mutant ◽  
Recycling Endosomes ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway

Electroneutral NaCl absorption in the ileum and colon is mediated by downregulated in adenoma (DRA) (Cl-/HCO3- exchanger; SLC26A3) and Na+/H+ exchanger 3 (NHE3, SLC9A3). Surface expression of transport proteins undergoes basal and regulated recycling by endo- and exocytosis. Expression and activity of DRA in the plasma membrane depend on intact lipid rafts, phosphatidylinositol 3-kinase (PI3-kinase), and the PDZ interaction of DRA. However, it is unknown how the PDZ interaction of DRA affects its trafficking to the cell surface. Therefore, the (re)cycling pathway of DRA was investigated in HEK cells stably expressing enhanced green fluorescent protein (EGFP)-DRA or EGFP-DRA-ETKFminus (a mutant lacking the PDZ interaction motif). Early, late, and recycling endosomes were immunoisolated by precipitating stably transfected mCherry-hemagglutinin (HA)-Rab5a, -7a, or -11a. EGFP-DRA and EGFP-DRA-ETKFminus were equally present in early endosomes. In recycling endosomes, wild-type DRA was preferentially present, whereas, in late endosomes, DRA-ETKF-minus dominated. Correspondingly, EGFP-DRA colocalized with mCherry-HA-Rab11a in recycling endosomes, whereas EGFP-DRA-ETKFminus colocalized with mCherry-HA-Rab7a in late endosomes. Functionally, this different distribution was reflected by a shorter half-life of the mutant DRA. Transient expression of dominant-negative Rab11aS25N inhibited the activity (-17%, P < 0.05) and the cell surface expression of DRA (-30%, P < 0.05). Transient transfection of Rab4a or its dominant-negative mutant Rab4aS22N was without effect and thus excluded participation of the rapid recycling pathway. Taken together, the PDZ interaction of DRA facilitates its movement into Rab11a-positive recycling endosomes, from where it is inserted in the plasma membrane. A scenario emerges where specific PDZ adaptor proteins are present along several compartments of the endocytosis-recycling pathway.

A SCL4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells

2004 ◽  
Vol 286 (3) ◽  
pp. C601-C610 ◽  
Author(s):  
J. Praetorius ◽  
L. N. Nejsum ◽  
S. Nielsen
Keyword(s):  
Cerebrospinal Fluid ◽  
Plasma Membrane ◽  
Choroid Plexus ◽  
Gene Product ◽  
Pcr Analysis ◽  
Membrane Domain ◽  
Choroid Plexus Epithelium ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
The Brain

The choroid plexus epithelium of the brain ventricular system produces the majority of the cerebrospinal fluid and thereby defines the ionic composition of the interstitial fluid in the brain. The transepithelial movement of Na+ and water in the choroid plexus depend on a yet-unidentified basolateral stilbene-sensitive [Formula: see text]-[Formula: see text] uptake protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression in the choroid plexus of SLC4A10 mRNA, which encodes a stilbene-sensitive [Formula: see text]-[Formula: see text] transporter. Anti-COOH-terminal antibodies were developed to determine the specific expression and localization of this [Formula: see text]-[Formula: see text] transport protein. Immunoblotting demonstrated antibody binding to a 180-kDa protein band from mouse and rat brain preparations enriched with choroid plexus. The immunoreactive band migrated as a 140-kDa protein after N-deglycosylation, consistent with the predicted molecular size of the SLC4A10 gene product. Bright-field immunohistochemistry and immunoelectron microscopy demonstrated strong labeling confined to the basolateral plasma membrane domain of the choroid plexus epithelium. Furthermore, the stilbene-insensitive [Formula: see text]-[Formula: see text] cotransporter, NBCn1, was also localized to the basolateral plasma membrane domain of the choroid plexus epithelium. Hence, we propose that the SLC4A10 gene product and NBCn1 both function as basolateral [Formula: see text] entry pathways and that the SLC4A10 gene product may be responsible for the stilbene-sensitive [Formula: see text]-[Formula: see text] uptake that is essential for cerebrospinal fluid production.

scholarly journals The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Annette Brandel ◽  
Sahaja Aigal ◽  
Simon Lagies ◽  
Manuel Schlimpert ◽  
Ana Valeria Meléndez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Plasma Membrane ◽  
Host Cell ◽  
Acyl Chain ◽  
Mass Spectrometry Analysis ◽  
Bacterial Invasion ◽  
Fatty Acyl Chain ◽  
Membrane Domain ◽  
Host Cell Membrane ◽  
Plasma Membrane Domain

AbstractThe opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP3 and flotillin thereby facilitating efficient uptake of PAO1.

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