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PubMed2000

Increased lysosome-related proteins in the skeletal muscles of distal myopathy with rimmed vacuoles.

Investigators have speculated that the degenerative process in distal myopathy with rimmed vacuoles (DMRV) mainly involves the lysosomal system. To investigate possible protein abnormalities related to intracellular lysosomal proteolytic pathways in DMRV-affected muscles, we performed immunohistochemical analyses of certain proteins in muscle biopsy specimens obtained from patients with various neuromuscular diseases, including DMRV, muscular dystrophy, polymyositis, and amyotrophic lateral sclerosis, and in normal human muscles specimens. Immunohistochemically, most muscle fibers in normal control specimens showed little or no reaction for clathrin and alpha- and gamma-subunits of adaptin-constituted adaptin proteins (AP)-1 and AP-2, respectively. Abnormal increases in these proteins were demonstrated mainly in the cytoplasm of atrophic fibers or in necrotic fibers in all diseased specimens. Particularly in DMRV-affected muscles, alpha- and gamma-adaptins were often observed inside or on the rims of vacuoles and in the cytoplasm of vacuolated fibers. Abnormal increases in Golgi-zone protein were also demonstrated in DMRV muscles. The rims of rimmed vacuoles were negative for kinectin, an endoplasmic reticulum-binding protein. Positive staining for both proteins, however, was sometimes seen inside the vacuoles in DMRV-affected fibers. These results suggest increased endocytosis at the plasma membrane as well as secretion involving transport from the trans-Golgi network of the Golgi apparatus in DMRV. Accumulation of various lysosome-related proteins within the rimmed vacuoles indicates at least some of these vacuoles may be autolysosomes.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis.

betaarrestins mediate the desensitization of the beta2-adrenergic receptor (beta2AR) and many other G protein-coupled receptors (GPCRs). Additionally, betaarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that betaarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, betaarrestin 2, and the beta2-adaptin subunit of AP-2. beta2-Adaptin binds betaarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with betaarrestins and beta2AR in an agonist-dependent manner in HEK-293 cells. Moreover, beta2-adaptin translocates from the cytosol to the plasma membrane in response to the beta2AR agonist isoproterenol and colocalizes with beta2AR in clathrin-coated pits. Finally, expression of betaarrestin 2 minigene constructs containing the beta2-adaptin interacting region inhibits beta2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

Adaptor Protein Complex alpha Subunits↗
PubMed1993

Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells.

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double-immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.

3T3 Cells↗
PubMed2001

Adaptins: the final recount.

Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles. In this article, we report the results of a survey for adaptins from sequenced genomes including those of man, mouse, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the plant Arabidopsis thaliana, and the yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We find that humans, mice, and Arabidopsis thaliana have four AP complexes (AP-1, AP-2, AP-3, and AP-4), whereas D. melanogaster, C. elegans, S. cerevisiae, and S. pombe have only three (AP-1, AP-2, and AP-3). Additional diversification of AP complexes arises from the existence of adaptin isoforms encoded by distinct genes or resulting from alternative splicing of mRNAs. We complete the assignment of adaptins to AP complexes and provide information on the chromosomal localization, exon-intron structure, and pseudogenes for the different adaptins. In addition, we discuss the structural and evolutionary relationships of the adaptins and the genetic analyses of their function. Finally, we extend our survey to adaptin-related proteins such as the GGAs and stonins, which contain domains homologous to the adaptins.

ADP-Ribosylation Factors↗
PubMed1997

Serine 331 and tyrosine 333 are both involved in the interaction between the cytosolic domain of TGN38 and the mu2 subunit of the AP2 clathrin adaptor complex.

TGN38 is a type I integral membrane protein that cycles between the trans-Golgi network and the plasma membrane. Internalization at the cell surface and targeting back to the trans-Golgi network is dependent on a hexapeptide motif, SDYQRL, in the cytosolic tail of the protein. It was recently demonstrated that this motif specifically interacts with the mu2 subunit of the AP2 adaptor complex. We have studied the interaction between the entire cytosolic domain of TGN38 and mu2 using the yeast two hybrid system, in vitro binding of recombinant fusion proteins and IAsys optical biosensor technology. A specific interaction has been demonstrated in each of the systems we have employed. We have shown an absolute requirement for Tyr-333 of TGN38 in binding to mu2. In addition we found that mutation of Ser-331 to alanine reduces the affinity of the interaction. By measuring tryptophan fluorescence at equilibrium, we have also determined the dissociation constant for the interaction between the entire cytosolic tail of TGN38 and mu2 as 58 nM. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the specificity of binding of TGN38 to mu2.

Adaptor Protein Complex 1↗
PubMed2001

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells.

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

Adaptor Protein Complex 1↗
PubMed1999

Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis.

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.

Adaptor Protein Complex 1↗
PubMed1999

Defective expression of the mu3 subunit of the AP-3 adaptor complex in the Drosophila pigmentation mutant carmine.

The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (mu) chain families mu1, mu2, and mu3, of the corresponding AP complexes, and delta-COP, the analogous component of the coatomer (COPI) complex. The Drosophila orthologs exhibit a high degree of sequence identity to mammalian medium chain and delta-COP proteins. Northern analysis demonstrated that medium chain and delta-COP mRNAs are expressed uniformly throughout fly development. Medium chain and delta-COP genes were cytologically mapped and the mu3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm1 mutant allele indicated the presence of a large insertion in the coding region of the mu3 gene and Northern analysis revealed no detectable mu3 mRNA. Light microscopy of the cm1 mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence of a role for mu3 in the sorting processes required for pigment granule biogenesis in Drosophila.

Adaptor Protein Complex 1↗
PubMed1997

Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation.

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.

Adaptor Protein Complex alpha Subunits↗
PubMed2000

Multiple C-terminal motifs of the 46-kDa mannose 6-phosphate receptor tail contribute to efficient binding of medium chains of AP-2 and AP-3.

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.

Adaptor Protein Complex 1↗
PubMed1992

The mechanism of receptor-mediated endocytosis: more questions than answers.

Receptor-mediated endocytosis occurs via clathrin-coated pits and is therefore coupled to the dynamic cycle of assembly and disassembly of the coat constituents. These coat proteins comprise part, but certainly not all, of the machinery involved in the recognition of membrane receptors and their selective packaging into transport vesicles for internalization. Despite considerable knowledge about the biochemistry of coated vesicles and purified coat proteins, little is known about the mechanisms of coated pit assembly, receptor-sorting and coated vesicle formation. Cell-free assays which faithfully reconstitute these events provide powerful new tools with which to elucidate the overall mechanism of receptor-mediated endocytosis.

Adaptor Protein Complex alpha Subunits↗
PubMed1992

Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators.

Brefeldin A (BFA) causes a rapid redistribution of coat proteins (e.g., gamma-adaptin) associated with the clathrin-coated vesicles that bud from the trans-Golgi network (TGN), while the clathrin-coated vesicles that bud from the plasma membrane are unaffected. gamma-Adaptin redistributes with the same kinetics as beta-COP, a coat protein associated with the non-clathrin-coated vesicles that bud from the Golgi complex. Upon removal of BFA, however, gamma-adaptin recovers its perinuclear distribution more rapidly. Redistribution of both proteins can be prevented by pretreating cells with AlF4-. Recruitment of adaptors from the cytosol onto the TGN membrane has been reconstituted in a permeabilized cell system and is increased by addition of GTP gamma S and blocked by addition of BFA. These results suggest a role for G proteins in the control of the clathrin-coated vesicle cycle at the TGN and further extend the similarities between clathrin-coated vesicles and non-clathrin-coated vesicles.

Adaptor Protein Complex alpha Subunits↗
PubMed1993

The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein.

The small GTP-binding protein, ADP-ribosylation factor (ARF), has previously been shown to mediate the binding to Golgi membranes of the coatomer of non-cathrin-coated (COP-coated) vesicles. We now report that ARF is also required for the binding of the AP-1 adaptor protein of clathrin-coated vesicles to Golgi membranes. The binding of coat proteins from both clathrin- and COP-coated vesicles requires an additional Golgi membrane-associated factor. These results suggest that a mechanistic similarity underlies diverse types of vesicle coats.

ADP-Ribosylation Factors↗
PubMed1999

Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells.

The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins. Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting. Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting. Here, we report the identification of a novel member of the adaptor medium chain family, named mu1B, which is closely related to the previously described mu1A (79% amino acid sequence identity). Northern blotting and in situ hybridization analyses reveal the specific expression of mu1B mRNA in a subset of polarized epithelial and exocrine cells. Yeast two-hybrid analyses show that mu1B is capable of interacting with generic tyrosine-based sorting signals. These observations suggest that mu1B may be involved in protein sorting events specific to polarized cells.

Adaptor Protein Complex 1↗
PubMed1998

A function for the AP3 coat complex in synaptic vesicle formation from endosomes.

Synaptic vesicles can be coated in vitro in a reaction that is ARF-, ATP-, and temperature-dependent and requires synaptic vesicle membrane proteins. The coat is largely made up of the heterotetrameric complex, adaptor protein 3, recently implicated in Golgi-to-vacuole traffic in yeast. Depletion of AP3 from brain cytosol inhibits small vesicle formation from PC12 endosomes in vitro. Budding from washed membranes can be reconstituted with purified AP3 and recombinant ARF1. We conclude that AP3 coating is involved in at least one pathway of small vesicle formation from endosomes.

ADP-Ribosylation Factor 1↗
PubMed1998

Clathrin and adaptors.

Clathrin and adaptors are components of clathrin-coated pits and vesicles. The AP-1 adaptor complex is associated with clathrin-coated vesicles budding from the TGN, while the AP-2 adaptor complex is associated with clathrin-coated vesicles budding from the plasma membrane. The clathrin forms a polyhedral lattice and is believed to be the driving force behind membrane invagination leading to vesicle budding. The adaptors attach the clathrin to the membrane and also interact with the cytoplasmic domains of selected transmembrane proteins, causing these proteins to become concentrated in clathrin-coated vesicles. Clathrin-coated vesicles budding from the TGN have been implicated in the sorting of newly synthesised lysosomal enzymes, while clathrin-coated vesicles budding from the plasma membrane facilitate the receptor-mediated endocytosis of ligands, such as low density lipoproteins and transferrin. A novel adaptor-related complex, AP-3, has recently been identified, which is recruited onto membranes of the TGN and a more peripheral compartment but does not appear to be associated with clathrin. Genetic studies indicate that AP-3 plays a role in the sorting of proteins to lysosomes and lysosome-related organelles.

Adaptor Protein Complex 3↗
PubMed2004

Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2.

The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.

Adaptor Protein Complex alpha Subunits↗