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Variants at 21 genetic loci have been associated with an increased risk for Alzheimer's disease (AD). An important unresolved question is whether multiple genetic risk factors can be combined to increase the power to detect changes in neuroimaging biomarkers for AD. We acquired high-resolution structural images of the hippocampus in 66 healthy, older human subjects. For 45 of these subjects, longitudinal 2-year follow-up data were also available. We calculated an additive AD genetic risk score for each participant and contrasted this with a weighted risk score (WRS) approach. Each score included APOE (apolipoprotein E), CLU (clusterin), PICALM (phosphatidylinositol binding clathrin assembly protein), and family history of AD. Both unweighted risk score (URS) and WRS correlated strongly with the percentage change in thickness across the whole hippocampal complex (URS: r = -0.40; p = 0.003; WRS: r = -0.25, p = 0.048), driven by a strong relationship to entorhinal cortex thinning (URS: r = -0.35; p = 0.009; WRS: r = -0.35, p = 0.009). By contrast, at baseline the risk scores showed no relationship to thickness in any hippocampal complex subregion. These results provide compelling evidence that polygenic AD risk scores may be especially sensitive to structural change over time in regions affected early in AD, like the hippocampus and adjacent entorhinal cortex. This work also supports the paradigm of studying genetic risk for disease in healthy volunteers. Together, these findings will inform clinical trial design by supporting the idea that genetic prescreening in healthy control subjects can be useful to maximize the ability to detect an effect on a longitudinal neuroimaging endpoint, like hippocampal complex cortical thickness.

Phosphatidylinositolbinding clathrin assembly protein (PICALM) gene is one novel genetic player associated with late-onset Alzheimer's disease (LOAD), based on recent genome wide association studies (GWAS). However, how it affects AD occurrence is still unknown. Brain reserve hypothesis highlights the tolerant capacities of brain as a passive means to fight against neurodegenerations. Here, we took the baseline volume and/or thickness of LOAD-associated brain regions as proxies of brain reserve capacities and investigated whether PICALM genetic variations can influence the baseline reserve capacities and the longitudinal atrophy rate of these specific regions using data from Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset. In mixed population, we found that brain region significantly affected by PICALM genetic variations was majorly restricted to posterior cingulate. In sub-population analysis, we found that one PICALM variation (C allele of rs642949) was associated with larger baseline thickness of posterior cingulate in health. We found seven variations in health and two variations (rs543293 and rs592297) in individuals with mild cognitive impairment were associated with slower atrophy rate of posterior cingulate. Our study provided preliminary evidences supporting that PICALM variations render protections by facilitating reserve capacities of posterior cingulate in non-demented elderly.

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer's disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.

Familial subvalvular aortic stenosis (SAS) is one of the most common congenital heart defects in dogs and is an inherited defect of Newfoundlands, golden retrievers and human children. Although SAS is known to be inherited, specific genes involved in Newfoundlands with SAS have not been defined. We hypothesized that SAS in Newfoundlands is inherited in an autosomal dominant pattern and caused by a single genetic variant. We studied 93 prospectively recruited Newfoundland dogs, and 180 control dogs of 30 breeds. By providing cardiac screening evaluations for Newfoundlands we conducted a pedigree evaluation, genome-wide association study and RNA sequence analysis to identify a proposed pattern of inheritance and genetic loci associated with the development of SAS. We identified a three-nucleotide exonic insertion in phosphatidylinositol-binding clathrin assembly protein (PICALM) that is associated with the development of SAS in Newfoundlands. Pedigree evaluation best supported an autosomal dominant pattern of inheritance and provided evidence that equivocally affected individuals may pass on SAS in their progeny. Immunohistochemistry demonstrated the presence of PICALM in the canine myocardium and area of the subvalvular ridge. Additionally, small molecule inhibition of clathrin-mediated endocytosis resulted in developmental abnormalities within the outflow tract (OFT) of Xenopus laevis embryos. The ability to test for presence of this PICALM insertion may impact dog-breeding decisions and facilitate reduction of SAS disease prevalence in Newfoundland dogs. Understanding the role of PICALM in OFT development may aid in future molecular and genetic investigations into other congenital heart defects of various species. © 2014 The Author(s).

The mouse hypopigmentation mutant pearl is an established model for Hermansky-Pudlak syndrome (HPS), a genetically heterogenous disease with misregulation of the biogenesis/function of melanosomes, lysosomes, and platelet dense granules. The pearl (Ap3b1) gene encodes the beta3A subunit of the AP-3 adaptor complex, which regulates vesicular trafficking. The genomic structure of the normal Ap3b1 gene includes 25 introns and a putative promoter sequence. The original pearl (pe) mutation, which has an unusually high reversion rate on certain strain backgrounds, has been postulated to be caused by insertion of a transposable element. Indeed, the mutation contains a 215-bp partial mouse transposon at the junction point of a large tandem genomic duplication of 6 exons and associated introns. At the cDNA level, three pearl mutations (pearl, pearl-8J, and pearl-9J) are caused by deletions or duplications of a complete exon(s).

The clinical significance and underlying molecular mechanism of miRNA-222-3p in metastatic prostate cancer (MPCa) remain unclear. The present study used a large number of cases (n = 1,502) based on miRNA chip and miRNA sequencing datasets to evaluate the expression and diagnostic potential of miRNA-222-3p in MPCa. We applied a variety of meta-analytic methods, including forest maps, sensitivity analysis, subgroup analysis and summary receiver operating characteristic curves, to prove the final results. MiRNA-222-3p was reduced in MPCa and had a moderate diagnostic potential in MPCa. We screened 118 miRNA-222-3p targets using three different methods including miRNA-222-3p transfected MPCa cell lines, online prediction databases and differently upregulated genes in MPCa. Moreover, functional enrichment analysis performed to explore the potential molecular mechanism of miRNA-222-3p showed that the potential target genes of miRNA-222-3p were significantly enriched in the p53 signal pathway. In the protein-protein interaction network analysis, SNAP91 was identified as a hub gene that may be closely related to MPCa. Gene chip and RNA sequencing datasets containing 1,237 samples were used to determine the expression level and diagnostic potential of SNAP91 in MPCa. SNAP91 was found to be overexpressed in MPCa and had a moderate diagnostic potential in MPCa. In addition, miRNA-222-3p expression was negatively correlated with SNAP91 expression in MPCa (r = -0.636, P = 0.006). These results demonstrated that miRNA-222-3p might play an important role in MPCa by negatively regulating SNAP91 expression. Thus, miRNA-222-3p might be a potential biomarker and therapeutic target of MPCa.

Phosphatidylinositol binding clathrin assembly protein (PICALM), also known as clathrin assembly lymphoid myeloid leukemia protein (CALM), was originally isolated as part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11)(p13;q14). CALM is sufficient to drive clathrin assembly in vitro on lipid monolayers and regulates clathrin-coated budding and the size and shape of the vesicles at the plasma membrane. However, the physiological role of CALM has yet to be elucidated. Here, the role of CALM in vivo was investigated using CALM-deficient mice. CALM-deficient mice exhibited retarded growth in utero and were dwarfed throughout their shortened life-spans. Moreover, CALM-deficient mice suffered from severe anemia, and the maturation and iron content in erythroid precursors were severely impaired. CALM-deficient erythroid cells and embryonic fibroblasts exhibited impaired clathrin-mediated endocytosis of transferrin. These results indicate that CALM is required for erythroid maturation and transferrin internalization in mice.

Clathrin assembly into coated pits and vesicles is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. These interactions may be mimicked in vitro by the assembly and disassembly of clathrin "baskets." The chimera C58J is a minimal construct capable of supporting both reactions; it consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J domain of auxilin, which recruits Hsc70 to baskets. We studied the process of disassembly by using cryo-electron microscopy to identify the initial binding site of Hsc70 on clathrin-C58J baskets at pH 6, under which conditions disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs and (ii) there is another interaction at a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J domain of C58J. We propose that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub.

Regulated delivery and removal of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.

Synaptic vesicles (SVs) assemble at the presynaptic compartment through a clathrin-dependent mechanism that involves one or more assembly proteins (APs). The assembly protein AP180 is especially efficient at facilitating clathrin cage formation, but its precise ultrastructural localization in neurons is unknown. Using immunoelectron microscopy, we demonstrate the presynaptic localization of AP180 in axon terminals of rat cerebellar neurons. In contrast, the assembly protein AP2 was associated with both the presynaptic plasma membrane and the cytosolic side of the membrane at postsynaptic and extrasynaptic sites. Furthermore, ultrastructural analysis of primate retina showed that AP180 immunoreactivity was preferentially and highly enriched at ribbon synapses, where glutamate is released tonically at high levels and rapid vesicle turnover is essential. To maintain functional synaptic transmission, neurotransmitter-filled SVs must be readily available, and this requires proper reassembly of new vesicles. The expression of AP180, in addition to AP-2, in the clathrin-mediated endocytic pathway might add another level of control to SV reformation for efficient assembly of clathrin, effectively controlling the size of assembled vesicles and faithfully recovering SV-specific components., (Copyright 2002 Wiley-Liss, Inc.)

Endocytosed microbial antigens are primarily delivered to lysosomal compartments where antigen binding to MHC and CD1 molecules occurs in an acidic and proteolytically active environment. Signal-dependent delivery to lysosomes has been suggested for these antigen-presenting molecules, but molecular interactions with vesicular coat proteins and adaptors that direct their lysosomal sorting are poorly understood. Here CD1b but not other CD1 isoforms bound the AP-3 adaptor protein complex. In AP-3-deficient cells derived from patients with Hermansky-Pudlak syndrome type 2 (HPS-2), CD1b failed to efficiently gain access to lysosomes, resulting in a profound defect in antigen presentation. Since MHC class II traffics normally in AP-3-deficient cells, defects in CD1b antigen presentation may account for recurrent bacterial infections in HPS-2 patients.

We have used a site-specific photo-cross-linking approach to identify direct interactions between clathrin adaptor protein (AP)1 complexes and small GTPases of the ADP-ribosylation factor (ARF) family and to explore the specificity of this interaction on immature secretory granule (ISG) membranes. ISG membranes are a well-characterized, highly enriched preparation of membranes that has previously been shown to have the membrane-associated factors for ARF1 recruitment that are not present on artificial liposomes. All three classes of ARF proteins could be recruited to ISG membranes, displaying differential requirements for GTPgammaS. We found that ARF1, ARF5, and ARF6 interacted directly with the beta1-adaptin subunit of AP-1 in the presence of GTPgammaS. Furthermore, we observed a direct interaction between the switch 1 region of ARF1 and the N-terminal trunk domains of gamma- and beta1-adaptin. In addition, both ARF1 and ARF6 but not ARF5 interacted directly with the beta3- and delta-adaptin subunits of AP-3. No interaction was observed between AP-2 and any of the ARF proteins. Our results delineate the specificity and provide evidence of a direct interaction between different ARF proteins and the AP complexes AP-1 and AP-3 on natural ISG membranes and show that residues in the switch 1 region of ARF proteins can selectively bind to the trunk domains of these complexes.

Adaptor protein (AP) complexes are heterotetrameric assemblies of subunits named adaptins. Four AP complexes, termed AP-1, AP-2, AP-3, and AP-4, have been described in various eukaryotic organisms. Biochemical and morphological evidence indicates that AP complexes play roles in the formation of vesicular transport intermediates and the selection of cargo molecules for inclusion into these intermediates. This understanding is being expanded by the application of genetic interference procedures. Here, we review recent progress in the genetic analysis of the function of AP complexes, focusing on studies that make use of targeted interference or naturally-occurring mutations in various model organisms.

Epsin and AP180/CALM are important endocytic accessory proteins that are believed to be involved in the formation of clathrin coats. Both proteins associate with phosphorylated membrane inositol lipids through their epsin N-terminal homology domains and with other components of the endocytic machinery through short peptide motifs in their carboxyl-terminal segments. Using hydrodynamic and spectroscopic methods, we demonstrate that the parts of epsin 1 and AP180 that are involved in protein-protein interactions behave as poorly structured flexible polypeptide chains with little or no conventional secondary structure. The predominant cytosolic forms of both proteins are monomers. Furthermore, we show that recombinant epsin 1, like AP180, drives in vitro assembly of clathrin cages. We conclude that the epsin N-terminal homology domain-containing proteins AP180/CALM and epsin 1 have a very similar molecular architecture that is designed for the rapid and efficient recruitment of the principal coat components clathrin and AP-2 at the sites of coated pit assembly.

Adaptors are heterotetrameric complexes that mediate the incorporation of cargo into transport vesicles by interacting with sorting signals present in the cytosolic domain of transmembrane proteins. Four adaptors, AP-1 (beta 1, gamma, mu 1A or mu 1B, sigma 1), AP-2 (beta 2, alpha, mu 2, sigma 2), AP-3 (beta 3 , delta, mu 3, sigma 3) or AP-4 (beta 4, epsilon, mu 4, sigma 4), have been characterized. AP-1 and AP-3 mediate sorting events at the level of the TGN and/or endosomes, whereas AP-2 functions in endocytic clathrin coated vesicle formation; no function is known so far for AP-4. Here, we show that AP-4 can bind different types of cytosolic signals known to mediate basolateral transport in epithelial cells. Furthermore, in MDCK cells with depleted mu 4 protein levels, several basolateral proteins are mis-sorted to the apical surface, showing that AP-4 participates in basolateral sorting in epithelial cells.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disease consisting of oculocutaneous albinism and a storage pool deficiency resulting from absent platelet dense bodies. The disorder is genetically heterogeneous. The majority of patients, including members of a large genetic isolate in northwest Puerto Rico, have mutations in HPS1. Another gene, ADTB3A, was shown to cause HPS-2 in two brothers having compound heterozygous mutations that allowed for residual production of the gene product, the beta3A subunit of adaptor complex-3 (AP-3). This heterotetrameric complex serves as a coat protein-mediating formation of intracellular vesicles, e.g. the melanosome and platelet dense body, from membranes of the trans-Golgi network. We determined the genomic organization of the human ADTB3A gene, with intron/exon boundaries, and describe a third patient with beta3A deficiency. This 5-y-old boy has two nonsense mutations, C1578T (R-->X) and G2028T (E-->X), which produce no ADTB3A mRNA and no beta3A protein. The associated mu3 subunit of AP-3 is also entirely absent. In fibroblasts, the cell biologic concomitant of this deficiency is robust and aberrant trafficking through the plasma membrane of LAMP-3, an integral lysosomal membrane protein normally carried directly to the lysosome. The clinical concomitant is a severe, G-CSF-responsive neutropenia in addition to oculocutaneous albinism and platelet storage pool deficiency. Our findings expand the molecular, cellular, and clinical spectrum of HPS-2 and call for an increased index of suspicion for this diagnosis among patients with features of albinism, bleeding, and neutropenia.

The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

The role of phospholipase Cgamma1 (PLCgamma1) in signal transduction was investigated by characterizing its SH domain-binding proteins that may represent components of a novel signaling pathway. A 180-kDa protein that binds to the SH2 domain of PLCgamma1 was purified from rat brain. The amino acid sequence of peptide derived from the purified protein is now identified as AP180, a clathrin assembly protein that has been implicated in clathrin-mediated synaptic vesicle recycling in synapses. In this report, we demonstrate the stable association of PLCgamma1 with AP180 in a clathrin-coated vesicle complex, which not only binds to the carboxyl-terminal SH2 domain of PLCgamma1, but also inhibits its enzymatic activity in a dose-dependent manner., ((c)2002 Elsevier Science.)

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.

Heterotetrameric adaptor complexes vesiculate donor membranes. One of the adaptor protein complexes, AP-3, is present in two forms; one form is expressed in all tissues of the body, whereas the other is restricted to brain. Mice lacking both the ubiquitous and neuronal forms of AP-3 exhibit neurological disorders that are not observed in mice that are mutant only in the ubiquitous form. To begin to understand the role of neuronal AP-3 in neurological disease, we investigated its function in in vitro assays as well as its localization in neural tissue. In the presence of GTPgammaS both ubiquitous and neuronal forms of AP-3 can bind to purified synaptic vesicles. However, only the neuronal form of AP-3 can produce synaptic vesicles from endosomes in vitro. We also identified that the expression of neuronal AP-3 is limited to varicosities of neuronal-like processes and is expressed in most axons of the brain. Although the AP-2/clathrin pathway is the major route of vesicle production and the relatively minor neuronal AP-3 pathway is not necessary for viability, the absence of the latter could lead to the neurological abnormalities seen in mice lacking the expression of AP-3 in brain. In this study we have identified the first brain-specific function for a neuronal adaptor complex.

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.

The Quail Neuroretina clone 71 gene (QNR-71) is expressed during the differentiation of retinal pigmented epithelia and the epidermis. It encodes a type I transmembrane glycoprotein that shares significant sequence homologies with several melanosomal proteins. We have studied its intracellular traffic in both pigmented and non-pigmented cells. We report that a di-leucine-based sorting signal (ExxPLL) present in the cytoplasmic domain of QNR-71 is necessary and sufficient for its proper targeting to the endosomal/premelanosomal compartments of both pigmented and non-pigmented cells. The intracellular transport of QNR-71 to these compartments is mediated by the AP-3 assembly proteins. As previously observed for the lysosomal glycoproteins Lampl and LimpII, overexpression of QNR-71 increases the amount of AP-3 associated with membranes, and inhibition of AP-3 synthesis increases the routing of QNR-71 towards the cell surface. In addition, expression of QNR-71 induces a misrouting of endogenous LampI to the cell surface. Thus, the targeting of QNR-71 might be similar to that of the lysosomal integral membrane glycoproteins LampI and LimpII. This suggests that sorting to melanosomes and lysosomes requires similar sorting signals and transport machineries.

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and alpha-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

MHC class II-restricted antigen presentation requires trafficking of newly synthesized class II-invariant chain complexes from the trans-Golgi network to endosomal, peptide-loading compartments. This transport is mediated by dileucine-like motifs within the cytosolic tail of the invariant chain. Although these signals have been well characterized, the cytosolic proteins that interact with these dileucine signals and mediate Golgi sorting and endosomal transport have not been identified. Recently, an adaptor complex, AP-3, has been identified that interacts with dileucine motifs and mediates endosomal/lysosomal transport in yeast, Drosophila, and mammals. In this report, we have assessed class II-invariant chain trafficking in a strain of mice (mocha) which lacks expression of AP-3. Our studies demonstrate that the lack of AP-3 does not affect the kinetics of invariant chain degradation, the route of class II-invariant chain transport, or the rate and extent of class II-peptide binding as assessed by the generation of SDS-stable dimers. The possible role of other known or unknown adaptor complexes in class II-invariant chain transport is discussed., (Copyright 2001 Academic Press.)

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.

The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.

Clathrin-mediated endocytosis plays a major role in retrieving synaptic vesicles from the plasma membrane following exocytosis. This endocytic process requires AP180 (or a homolog), which promotes the assembly and restricts the size of clathrin-coated vesicles. The highly conserved 33 kDa amino-terminal domain of AP180 plays a critical role in binding to phosphoinositides and in regulating the clathrin assembly activity of AP180. The crystal structure of the amino-terminal domain reported herein reveals a novel fold consisting of a large double layer of sheets of ten alpha helices and a unique site for binding phosphoinositides. The finding that the clathrin-box motif is mostly buried and lies in a helix indicates a different site and mechanism for binding of the domain to clathrins than previously assumed.

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.

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.

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

Dynamin I and at least five other nerve terminal proteins, amphiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and amphiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes of cno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3-independent mechanism critical to the biogenesis of lysosome-related organelles. (Blood. 2000;96:4227-4235)

Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.

There are several adaptor proteins associated with clathrin coated vesicles. Among them are AP180 and AP-2. We and others have previously described synaptic localization of AP180. AP180 immunoreactivity is altered in both the superior frontal gyrus and hippocampus in Alzheimer's disease (AD). We here investigate the location and alteration of another adaptor protein, AP-2. In contrast to AP180, we have found that AP-2 is expressed by both neurons and glia. Furthermore, the only noticeable change of AP-2 in AD is a loss of its immunoreactivity in layer II of the superior frontal gyrus.

The heterotetrameric adaptor protein complex AP-3 has been shown to function in the sorting of proteins to the endosomal/lysosomal system. However, the mechanism of AP-3 recruitment onto membranes is poorly understood, and it is still uncertain whether AP-3 nucleates clathrin-coated vesicles. Using purified components, we show that AP-3 and clathrin are recruited onto protein-free liposomes and Golgi-enriched membranes by a process that requires ADP-ribosylation factor (ARF) and GTP but no other proteins or nucleotides. The efficiency of recruitment onto the two sources of membranes is comparable and independent of the composition of the liposomes. Clathrin binding occurred in a cooperative manner as a function of the membrane concentration of AP-3. Thin-section electron microscopy of liposomes and Golgi-enriched membranes that had been incubated with AP-3, clathrin, and ARF.GTP showed the presence of clathrin-coated buds and vesicles. These results establish that AP-3-containing clathrin-coated vesicles form in vitro and are consistent with AP-3-dependent protein transport being mediated by clathrin-coated vesicles.

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

The AP-3 adaptor protein complex has been implicated in the biogenesis of lysosome-related organelles, such as pigment granules/melanosomes, and synaptic vesicles. Here we compare the relative importance of AP-3 in the biogenesis of these organelles in Drosophila melanogaster. We report that the Drosophila pigmentation mutants orange and ruby carry genetic lesions in the sigma3 and beta3-adaptin subunits of the AP-3 complex, respectively. Electron microscopy reveals dramatic reductions in the numbers of electron-dense pigment granules in the eyes of these AP-3 mutants. Mutant flies also display greatly reduced levels of pigments housed in these granules. In contrast, electron microscopy of retinula cells reveals numerous synaptic vesicles in both AP-3 mutant and wild-type flies, while behavioral assays show apparently normal locomotor ability of AP-3 mutant larvae. Together, these results demonstrate that Drosophila AP-3 is critical for the biogenesis of pigment granules, but is apparently not essential for formation of a major population of synaptic vesicles in vivo.

Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein., (Copyright 2000 Academic Press.)

The major histocompatibility complex class II subunits (MHC-II) alpha and beta assemble with the invariant chain (Ii) in the endoplasmic reticulum and are transported to endosomal-lysosomal organelles known as MHC class II compartments (MIICs). Although it has been shown that two dileucine-based signals in the cytosolic tail of Ii, as well as a dileucine-based signal in the tail of the beta chain mediate sorting to MIICs, the molecular mechanisms by which alphabetaIi complexes are sorted have yet to be resolved fully. The AP-3 adaptor complex stands out as a particularly good candidate for mediating this targeting because: (i) it has a proven role in the trafficking of membrane proteins to lysosome-related organelles; and (ii) it has the ability to interact with dileucine-based signals in vitro. To investigate the potential role of AP-3 in transport of MHC-II to MIICs, we have examined MHC-II trafficking in human B-lymphoblast lines from patients with Hermansky-Pudlak syndrome type 2 (HPS-2), which are deficient in the AP-3 complex. Pulse-chase analyses revealed no significant alteration in the kinetics of synthesis and degradation of either MHC-II subunits or Ii. Moreover, we observed neither impairment of the formation of compact SDS-resistant alphabeta dimers, nor delay in the appearance of a conformational epitope indicative of a mature, Ii-free alphabeta dimer. Finally, we demonstrated that in HPS-2 patients' cells, there was no delay in the expression of the alphabeta dimers on the cell surface. Thus, AP-3 does not seem to be essential for normal trafficking of MHC-II. These findings have important implications for HPS-2 patients, because they suggest that the recurrent bacterial infections suffered by these patients are not likely due to impaired antigen processing and presentation by MHC-II.

Lysosomal protein trafficking is a fundamental process conserved from yeast to humans. This conservation extends to lysosome-like organelles such as mammalian melanosomes and insect eye pigment granules. Recently, eye and coat color mutations in mouse (mocha and pearl) and Drosophila (garnet and carmine) were shown to affect subunits of the heterotetrameric adaptor protein complex AP-3 involved in vesicle trafficking. Here we demonstrate that the Drosophila eye color mutant ruby is defective in the AP-3beta subunit gene. ruby expression was found in retinal pigment and photoreceptor cells and in the developing central nervous system. ruby mutations lead to a decreased number and altered size of pigment granules in various cell types in and adjacent to the retina. Humans with lesions in the related AP-3betaA gene suffer from Hermansky-Pudlak syndrome, which is caused by defects in a number of lysosome-related organelles. Hermansky-Pudlak patients have a reduced skin pigmentation and suffer from internal bleeding, pulmonary fibrosis, and visual system malfunction. The Drosophila AP-3beta adaptin also appears to be involved in processes other than eye pigment granule biogenesis because all ruby allele combinations tested exhibited defective behavior in a visual fixation paradigm.

The "WD40" domain is a widespread recognition module for linking partner proteins in intracellular networks of signaling and sorting. The clathrin amino-terminal domain, which directs incorporation of cargo into coated pits, is a beta-propeller closely related in structure to WD40 modules. The crystallographically determined structures of complexes of the clathrin-terminal domain with peptides derived from two different cargo adaptors, beta-arrestin 2 and the beta-subunit of the AP-3 complex, reveal strikingly similar peptide-in-groove interactions. The two peptides in our structures contain related, five-residue motifs, which form the core of their contact with clathrin. A number of other proteins involved in endocytosis have similar "clathrin-box" motifs, and it therefore is likely that they all bind the terminal domain in the same way. We propose that a peptide-in-groove interaction is an important general mode by which beta-propellers recognize specific target proteins.

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.