The isolation of clathrin-coated vesicles and purification of their protein components.
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Adaptor Protein Complex alpha Subunits↗
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Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis.
The Drosophila dicistronic stoned locus encodes two distinctive presynaptic proteins, Stoned A (STNA) and Stoned B (STNB); STNA is a novel protein without homology to known synaptic proteins, and STNB contains a domain with homology to the endocytotic protein AP50. Both Stoned proteins colocalize precisely with endocytotic proteins including the AP2 complex and Dynamin in the "lattice network" characteristic of endocytotic domains in Drosophila presynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. We have documented previously a highly specific synaptic mislocalization and degradation of Synaptotagmin I in stoned mutants. Here we show that transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. We conclude that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane.
Adaptor Protein Complex 2↗
The adaptor complexes: a bridge between the transmembrane proteins and clathrin lattices.
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Acid Phosphatase↗
Functional analysis of AP-2 alpha and mu2 subunits.
The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of alpha, or mutating the PtdIns(4,5)P2 binding site of mu2, has no apparent effect. However, adding a C-terminal GFP tag to alpha renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo.
Adaptor Protein Complex alpha Subunits↗
HIV and SIV Nef modulate signal transduction and protein sorting in T cells.
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Adaptor Protein Complex alpha Subunits↗
A structural explanation for the binding of multiple ligands by the alpha-adaptin appendage domain.
The alpha subunit of the endocytotic AP2 adaptor complex contains a 30 kDa "appendage" domain, which is joined to the rest of the protein via a flexible linker. The 1.9 A resolution crystal structure of this domain reveals a single binding site for its ligands, which include amphiphysin, Eps15, and epsin. This domain when overexpressed in COS7 fibroblasts is shown to inhibit transferrin uptake, whereas mutants in which interactions with its binding partners are abolished do not. DPF/W motifs present in appendage domain-binding partners are shown to play a crucial role in their interactions with the domain. A single site for binding multiple ligands would allow for temporal and spatial regulation in the recruitment of components of the endocytic machinery.
Adaptor Protein Complex 2↗
Different domains of the AP-1 adaptor complex are required for Golgi membrane binding and clathrin recruitment.
The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the beta 1 and gamma subunits and associated mu 1 and sigma 1 subunits, and lacking either the beta 1 subunit carboxyl-terminal appendage or both beta 1 and gamma subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTP gamma S. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the beta 1 and gamma subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the beta 1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the beta 1 and gamma subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact beta 1 subunit.
3T3 Cells↗
Regulation and function of the CD3gamma DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro.
Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3gamma. Thus, phosphorylation of CD3gamma serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3gamma internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3gamma molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3gamma S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.
Adaptor Protein Complex alpha Subunits↗
Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites.
Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.
Adaptor Protein Complex alpha Subunits↗
Purification of Golgi adaptor protein 1 from bovine adrenal gland and characterization of its beta 1 (beta') subunit by microsequencing.
A method for the purification of the Golgi adaptor protein 1 from bovine adrenal gland tissue was devised to investigate the relationship of its beta 1 (formerly referred to as beta') subunit to known beta-type sequences. Adrenal gland tissue was chosen for this study because it yielded 2-3 times more adaptor protein 1 than a comparable preparation from bovine brain. Like its neuronal isoform, the beta 1 subunit from adrenal gland adaptor protein 1 is readily cleaved by trypsin into a 63-kDa N-terminal fragment and a 40-kDa C-terminal fragment, while the gamma subunit is largely refractory to digestion. Based on microsequencing of 167 residues from the 63-kDa fragment, we noted 11 differences to the corresponding region of the beta 2 (formerly beta) subunit of the plasma membrane adaptor protein 2, but only one difference to the corresponding region of a beta-type protein encoded by the rat cDNA clone AP105a which is supposed to be a variant of the beta 2 subunit of the plasma membrane adaptor protein 2 [Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. P., Burne, C., Keen, J. H. & Davis, A. E. (1989) Proc. Natl Acad. Sci. USA 84, 8805-8809]. Alignment of 187 residues from the 40-kDa beta 1 C-terminal fragment revealed differences in 77 positions to the corresponding region of the beta 2 subunit and differences in 23 positions compared to the supposed beta 2-like protein. These findings suggest that the protein encoded by the rat cDNA clone AP105a is more closely related to the beta 1 subunit of the bovine adrenal Golgi adaptor protein 1 than to the beta 2 subunit of the rat plasma membrane adaptor protein 2.
Adaptor Protein Complex 1↗
COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP.
We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two-hybrid system, we characterize the domain structure of the alpha-, beta'-, epsilon-COP and beta-, gamma-, delta-, zeta-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the beta-, gamma-, delta-, zeta-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of alpha-COP truncation mutants, we characterize distinct functional domains on alpha-COP. Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent trafficking. The last approximately 170 amino acids of alpha-COP are also non-essential for cell viability, but required for epsilon-COP incorporation into coatomer and maintainance of normal epsilon-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: beta'- and gamma-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and beta- and epsilon-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.
ADP-Ribosylation Factors↗
Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression.
Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.
Adaptor Protein Complex alpha Subunits↗
Coat proteins regulating membrane traffic.
This review focuses on the roles of coat proteins in regulating the membrane traffic of eukaryotic cells. Coat proteins are recruited to the donor organelle membrane from a cytosolic pool by specific small GTP-binding proteins and are required for the budding of coated vesicles. This review first describes the four types of coat complexes that have been characterized so far: clathrin and its adaptors, the adaptor-related AP-3 complex, COPI, and COPII. It then discusses the ascribed functions of coat proteins in vesicular transport, including the physical deformation of the membrane into a bud, the selection of cargo, and the targeting of the budded vesicle. It also mentions how the coat proteins may function in an alternative model for transport, namely via tubular connections, and how traffic is regulated. Finally, this review outlines the evidence that related coat proteins may regulate other steps of membrane traffic.
Adaptor Protein Complex alpha Subunits↗
A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals.
Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the alpha c subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis.
Adaptor Protein Complex 2↗
Reduction of dietary obesity in aP2-Ucp transgenic mice: mechanism and adipose tissue morphology.
C57BL6/J mice with the expression of the mitochondrial uncoupling protein (UCP) gene from the fat-specific aP2 gene promoter were used to study the mechanism by which the aP2-Ucp transgene affects adiposity and reduces high-fat diet induced obesity. In the transgenic mice, UCP synthesized in white fat was inserted into mitochondria, and oxygen uptake by epididymal fat fragments indicated UCP-induced thermogenesis. The respirometry data, UCP content, cytochrome oxidase activity, and tissue morphology suggested functional involution of brown fat. Despite 25- to 50-fold lower mitochondrial cytochrome oxidase activity in white than in brown fat cells, total oxidative capacity in white and brown adipose tissue is comparable. Appearance of novel small cells in the gonadal fat of the transgenic mice was associated with a higher DNA content than that of the nontransgenic mice. The results prove a potential of transgenically altered mitochondria in white fat to modulate adiposity and energy expenditure and suggest the existence of a yet unidentified site-specific link between energy metabolism in adipocytes and cellularity.
Adaptor Protein Complex 2↗
Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis.
The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.
Adaptor Protein Complex 2↗
Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits.
The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH(2)-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922-20929). Here we used deletion and site- directed mutagenesis to determine that alpha residues 21-80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55-57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated alpha subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871-881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits.
Adaptor Protein Complex alpha Subunits↗
Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2.
Disabled-2 (Dab2) is a widely expressed relative of Disabled-1, a neuron-specific signal-transduction protein that binds to and receives signals from members of the low-density lipoprotein receptor (LDLR) family. Members of the LDLR family internalize through clathrin-coated pits and vesicles to endosomes, from where they return to the cell surface through the secretory pathway. In this study, we show that the Dab2 phosphotyrosine-binding domain binds peptides containing the sequence FXN-PXY. This core sequence is found in the intracellular domains of LDLR family members and is important for receptor internalization. Dab2 transiently colocalizes with the LDLR in clathrin-coated pits, but is absent from endosomes and lysosomes. Dab2 is alternatively spliced and its localization depends on a region of the protein that contains two DPF motifs that are present in the p96 Dab2 protein and absent in the p67 splice variant. This region is sufficient to confer Dab2 binding to the alpha-adaptin subunit of the clathrin adaptor protein, AP-2. Overexpression of p96 but not of p67 Dab2 disrupts the localization of AP-2. These findings suggest that in addition to previously reported signal-transduction functions, Dab2 could also act as an adaptor protein that may regulate protein trafficking.
3T3 Cells↗