A Novel Assay to Demonstrate an Intersection of the Exocytic and Endocytic Pathways at Early Endosomes

Laird, Vivienne; Spiess, Martin

doi:10.1006/excr.2000.5006

Cited by 19 publications

(14 citation statements)

References 35 publications

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“…2 and 31), in agreement with the predominant endosomal localization of transfected 1B (29,32). However, studies over the past decade have shown that some newly synthesized plasma membrane proteins move from TGN to recycling endosomes (33)(34)(35)(36)(37)(38), suggesting that the latter could act as a common sorting site in both routes for at least some basolateral proteins (2,12,31).…”

supporting

confidence: 53%

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells

Gravotta

Deora

Perret

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

142

233

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The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). 1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that 1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.

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supporting

confidence: 53%

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells

Gravotta

Deora

Perret

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

142

233

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“…Thus, the possibility that Cx33 acts as a chaperone that alters the stability of the gap junction plaques by maintaining Cx43 in the early endosome before recycling to the plasma membrane or to other cytoplasmic components can be hypothesized. Altered trafficking of the Cx33/Cx43 complex to the plasma membrane could be also considered since it has been demonstrated that early/recycling endosomes can also be part of the exocytic pathway (Laird and Spiess, 2000). In addition, a role of Cx in processes independent of the formation of gap junctional channels has been suggested (Huang et al, 1998;Krutovskikh et al, 2000;Plotkin et al, 2002;Olbina and Eckhart, 2003).…”

Section: Discussionmentioning

confidence: 99%

Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration

Fiorini

Mograbi

Cronier

et al. 2004

Journal of Cell Science

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Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation.Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. Highresolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.

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“…The tyrosine motif of TGN38, also interacted with AP-2 adaptors in vitro (Ohno et al, 1995) but only weakly with AP-1 (Boll et al, 1996); yeast two-hybrid assays with 1 yielded variable results (Ohno et al, 1995Rapoport et al, 1997;Stephens et al, 1997;Stephens and Banting, 1998). There is evidence that at least some membrane proteins are transported from the TGN to the basolateral surface via endosomes rather than in a direct vesicular transport route to the plasma membrane (Futter et al, 1995;Leitinger et al, 1995;Laird and Spiess, 2000;Orzech et al, 2000). Together with the recent discovery of a 1 isoform (1B) involved in basolateral sorting (Fö lsch et al, 1999;Ohno et al, 1999), AP-1 adaptors are thus potentially involved in surface transport of basolateral proteins, including TGN38.…”

Section: Discussionmentioning

confidence: 99%

ARF1·GTP, Tyrosine-based Signals, and Phosphatidylinositol 4,5-Bisphosphate Constitute a Minimal Machinery to Recruit the AP-1 Clathrin Adaptor to Membranes

Crottet

Meyer

Rohrer

et al. 2002

MBoC

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At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTPanalogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation. INTRODUCTIONSorting of membrane proteins is generally mediated by cytosolic coats which serve the dual role of creating a scaffold to form coated buds and vesicles and of selectively concentrating cargo proteins by interacting with cytosolic signals. The best studied systems are COPI in intra-Golgi and Golgito-endoplasmic reticulum (ER) transport, COPII in ER-toGolgi transport, and clathrin with associated adaptor proteins in the formation of vesicles at the plasma membrane, the trans-Golgi network (TGN) and endosomes. There are different types of clathrin-associated adaptor proteins (APs), heterotetrameric complexes composed of two ϳ100-kDa adaptins, a ϳ50-kDa medium (), and a ϳ20-kDa small () chain (Robinson and Bonifacino, 2001). The adaptor complexes form the inner layer of the coat that specifies the site of coat formation and interacts with cargo molecules. AP-1 adaptors are primarily functional at the TGN generating vesicles destined for endosomes but have also been found on sorting endosomes and implicated in (basolateral) recycling to the plasma membrane (Futter et al., 1998). AP-2 adaptors are found at the plasma membrane to form coated vesicles for endocytosis. AP-3 adaptors are involved in lysosomal transport from the TGN or endosomes. The different adaptor complexes recognize similar tyrosine and dileucine signals in cargo molecules, and in many cases the same signals are recognized by several adaptor types (Bonifacino and Dell'Angelica, 1999;Heilker et al., 1999).Recruitment of the different coats to their specific membranes appears to involve common basic mechanisms. With the exception of AP-2/clathrin coats, all the coats mentioned above require small GTPases that are activated from their soluble GDP-bound to their membrane-associated GTPbound form by a guanine nucleotide exchange factor (GEF) at the correct membrane. For COPII coats in yeast, the GTPase Sar1p is activated by the GEF Sec12p in the ER membrane. In an ...

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A Novel Assay to Demonstrate an Intersection of the Exocytic and Endocytic Pathways at Early Endosomes

Cited by 19 publications

References 35 publications

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells

Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration

ARF1·GTP, Tyrosine-based Signals, and Phosphatidylinositol 4,5-Bisphosphate Constitute a Minimal Machinery to Recruit the AP-1 Clathrin Adaptor to Membranes

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