Sorting of membrane proteins is generally mediated by cytosolic coats, which create a scaffold to form coated buds and vesicles and to selectively concentrate cargo by interacting with cytosolic signals. The classical paradigm is the interaction between clathrin coats and associated adaptor proteins, which cluster receptors with characteristic tyrosine and dileucine motifs during endocytosis. Clathrin in association with different sets of adaptors is found in addition at the trans‐Golgi network and endosomes. Sequences similar to internalization signals also direct lysosomal and basolateral sorting, which implicates related clathrin‐adaptor coats in the respective sorting pathways. This review concentrates on the recognition of sorting signals by clathrin‐associated adaptor proteins, an area of significant recent progress due to new methodological and conceptual approaches.  BioEssays 21:558–567, 1999. © 1999 John Wiley & Sons, Inc.

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and ␥-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5. INTRODUCTIONEarly endosomes are a major sorting station for proteins and membranes in eukaryotic cells (Gruenberg, 2001;Maxfield and McGraw, 2004). They receive material from the cell surface by endocytosis and from the exocytic pathway via the trans-Golgi network (TGN), and they distribute it further to late endosomes and back to the TGN and the plasma membrane. Transport receptors like the transferrin receptor, the low-density lipoprotein (LDL) receptor, and the asialoglycoprotein (ASGP) receptor cycle continuously between the plasma membrane and early endosomes (Spiess, 1990;Trowbridge et al., 1993). Receptor-positive early endosomes can be subdivided into sorting endosomes and recycling endosomes (or endocytic recycling compartment, ERC). Primary endocytic vesicles fuse to sorting endosomes where ligands dissociate from their receptors because of a reduced internal pH. The receptors exit into tubular membranes that form recycling endosomes, whereas the ligands with the main fluid volume mature to late endosomes ("geometrybased sorting"; Maxfield and McGraw, 2004). There seem to be two main recycling pathways from early endosomes to the plasma membrane: a fast one directly from sorting endosomes and a slower one via recycling endosomes (Sheff et al., 1999;Hao and Maxfield, 2000;.The mechanisms to generate endosome-derived recycling vesicles are not clear, because there are seemingly contradictory findings. In general, formation of transport vesicles between organelles of the endocytic and secretory pathways requires cytosolic coat proteins to be recruited at the membrane of the donor compartment. Among the established coat complexes, clathr...

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 ...

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membranebound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RII␣ as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of ␤-COP onto Golgi membranes containing prebound ARF1.

Previous studies have shown that the mucin-type polypeptides GlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectin only when they are synthesized by the specialized high-endothelial venules (HEV) of lymph modes. Since sialylation, sulfation, and possibly fucosylation are required for generating recognition, we reasoned that other mucins known to have such components might also bind L-selectin. We show here that soluble mucins secreted by human colon carcinoma cells, as well as those derived from human bronchial mucus can bind to human L-selectin in a calcium-dependent manner. As with Gly-CAM-1 synthesized by lymph node HEV, alpha 2-3 linked sialic acids and sulfation seem to play a critical role in generating this L-selectin binding. In each case, only a subset of the mucin molecules is recognized by L-selectin. Binding is not destroyed by boiling, suggesting that recognition may be based primarily upon carbohydrate structures. Despite this, O-linked oligosaccharide chains released from these ligands by beta-elimination do not show any detectable binding to L-selectin. Following protease treatment of the ligands, binding persists in a subset of the resulting fragments, indicating that specific recognition is determined by certain regions of the original mucins. However, O-linked oligosaccharides released from the subset of non-binding mucin fragments do not show very different size and charge profiles compared to those that do bind. Furthermore, studies with polylactosamine-degrading endoglycosidases suggest that the core structures involved in generating binding can vary among the different ligands. Taken together, these data indicate that a single unique oligosaccharide structure may not be responsible for high-affinity binding. Rather, diverse mucins with sialylated, sulfated, fucosylated lactosamine-type O-linked oligosaccharides can generate high-affinity L-selectin ligands, but only when they present these chains in unique spacing and/or clustered combinations, presumably dictated by the polypeptide backbone.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corth-é sy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670 -33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1⅐GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1⅐GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting. INTRODUCTIONIntracellular transport between membrane compartments is initiated by the recruitment of cytosolic coat proteins, which perform several functions (Kirchhausen, 2000;Aridor and Traub, 2002). They select and concentrate cargo proteins, polymerize to form a lattice structure on the membrane surface, and deform the lipid bilayer to bud toward the cytosol. On completion of a coated vesicle, the coat components disassemble, allowing fusion with the target compartment. The three best characterized coats are coat protein (COP) I mediating intra-Golgi and Golgi-to-endoplasmic reticulum transport, COPII for vesicles derived from the endoplasmic reticulum, and clathrin with various associated adaptor proteins for pathways between the plasma membrane, endosomes, and the trans-Golgi network (Kirchhausen, 2000).In all systems (apparently even for clathrin-dependent endocytosis; Paleotti et al., 2005), coat recruitment is initiated by a small GTPase that is activated at the membrane by a guanine nucleotide exchange factor (GEF). The minimal requirements to form coats have been defined in vitro using chemically defined liposomes and purified coat components. The generation of COPI vesicles required the heteroheptameric coatomer complex and ADP-ribosylation factor 1 (Arf1) and was enhanced by acidic phospholipids or by lipid-anchored sorting signals (Spang et al., 1998;. COPII consists of two dimers that can be sequentially assembled on liposomes containing phosphoinositides. Sec23/24 is first targeted by Sar1⅐GTP to the membrane as a primer to recruit the second layer of Sec13/31 (Matsuoka et al., 1998a(Matsuoka et al., , 1998b. Clathrin coats are similarly composed of two layers (Robinson and Bonifacino, 2001). Typically, heterotetrameric adaptor...

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