AP-1A and AP-3A lysosomal sorting functions

Heterotetrameric adaptor-protein complexes AP-1A and AP-3A mediate protein sorting in post-Golgi vesicular transport. AP-1A and AP-3A have been localized to the trans -Golgi network, indicating a function in protein sorting at this compartment. AP-3A appears to mediate trans -Golgi network-to-lysosome and also endosome-to-lysosome protein sorting. AP-1A is thought to be required for both trans -Golgi network-to-endosome transport and endosome-to- trans -Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized γ-ear containing, ARF binding protein) adaptor proteins in trans -Golgi network to endosome protein transport has brought into question the long-discussed trans -Golgi network-to-endosome sorting function of AP-1A. Murine cytomegalovirus gp48 contains an unusual di-leucine-based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP-1A, AP-3A and both, to determine their sorting functions. We find that AP1-A and AP3-A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady-state.     

Adaptor protein complexes and intracellular transport

The AP (adaptor protein) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes; whereas AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers that mediate transport from a donor membrane to a target organellar membrane. AP complexes play important roles in maintaining the normal physiological function of eukaryotic cells. Dysfunction of AP complexes has been implicated in a variety of inherited disorders, including: MEDNIK (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis and keratodermia) syndrome, Fried syndrome, HPS (Hermansky–Pudlak syndrome) and HSP (hereditary spastic paraplegia).     

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.     

Adaptor protein complexes as the key regulators of protein sorting in the post-Golgi network

Adaptor protein (AP) complexes are cytosolic heterotetramers that mediate the sorting of membrane proteins in the secretory and endocytic pathways. AP complexes are involved in the formation of clathrin-coated vesicles (CCVs) by recruiting the scaffold protein, clathrin. AP complexes also play a pivotal role in the cargo selection by recognizing the sorting signals within the cytoplasmic tail of integral membrane proteins. Six distinct AP complexes have been identified. AP-2 mediates endocytosis from the plasma membrane, while AP-1, AP-3 and AP-4 play a role in the endosomal/lysosomal sorting pathways. Moreover, tissue-specific sorting events such as the basolateral sorting in polarized epithelial cells and the biogenesis of specialized organelles including melanosomes and synaptic vesicles are also regulated by members of AP complexes. The application of a variety of methodologies have gradually revealed the physiological role of AP complexes.     

Genetic analyses of adaptin function from yeast to mammals

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.     

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.     

The fifth adaptor protein complex

Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.     

Clathrin adaptor AP-2 is essential for early embryonal development

The heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 play key roles in transport vesicle formation and cargo sorting in post-Golgi trafficking pathways. Studies on cultured mammalian cells have shown that AP-2 mediates rapid endocytosis of a subset of plasma membrane receptors. To determine whether this function is essential in the context of a whole mammalian organism, we carried out targeted disruption of the gene encoding the mu2 subunit of AP-2 in the mouse. We found that mu2 heterozygous mutant mice were viable and had an apparently normal phenotype. In contrast, no mu2 homozygous mutant embryos were identified among blastocysts from intercrossed heterozygotes, indicating that mu2-deficient embryos die before day 3.5 postcoitus (E3.5). These results indicate that AP-2 is indispensable for early embryonic development, which might be due to its requirement for cell viability.     

Early embryonic death of mice deficient in gamma-adaptin

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the gamma-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. gamma-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free beta1- or micro1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.     

Intracellular transport of MHC class II and associated invariant chain in antigen presenting cells from AP-3-deficient mocha mice

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.     

The AP-3 clathrin-associated complex is essential for embryonic and larval development in Caenorhabditis elegans

The adaptor protein (AP) complexes are involved in membrane transport of many proteins. There are 3 AP complexes in C. elegans unlike mammals that have four. To study the biological functions of the AP-3 complexes of C. elegans, we sought homologues of the mouse and human genes that encode subunits of the AP-3 complexes by screening C. elegans genomic and EST sequences. We identified single copies of homologues of the m3, s3, b3 and d genes. The medium chain of AP-3 is encoded by a single gene in C. elegans but two different genes in mammals. Since there are no known mutations in these genes in C. elegans, we performed RNAi to assess their functions in development. RNAi of each of the genes caused embryonic and larval lethal phenotypes. APM-3 is expressed in most cells, particularly strongly in spermatheca and vulva. We conclude that the products of the C. elegans m3, s3, b3 and d genes are essential for embryogenesis and larval development.     

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.     

The beta 1 and beta 2 subunits of the AP complexes are the clathrin coat assembly components.

The beta 1 and beta 2 subunits are the closely-related large chains of the trans-Golgi network AP-1 and the plasma membrane AP-2 clathrin-associated protein complexes, respectively. Recombinant beta 1 and beta 2 subunits have been generated in Escherichia coli. It was found that, in the absence of all the other AP subunits, beta 1 and beta 2 interact with clathrin and drive the efficient assembly of clathrin coats. In addition, beta 2 subunits and AP complexes compete for the same clathrin binding site. The appearance of the clathrin/beta coats is the same as the barrel-shaped structures formed with native AP complexes. It is proposed that the principal function of the beta subunits is to initiate coat formation, while the remaining subunits of the AP complexes have other roles in coated pit and coated vesicle function.     

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.     

Sorting of the v-SNARE VAMP7 in Dictyostelium discoideum: a role for more than one Adaptor Protein (AP) complex

Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism.     

HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.     

Characterization of a Fourth Adaptor-related Protein Complex

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (epsilon, beta4, mu4, and sigma4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to approximately 10-20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The mu4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.     

Conservation and diversification of dileucine signal recognition by adaptor protein (AP) complex variants

The clathrin-associated, heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, and AP-3, recognize signals in the cytosolic domains of transmembrane proteins, leading to their sorting to endosomes, lysosomes, lysosome-related organelles, and/or the basolateral membrane of polarized epithelial cells. One type of signal, referred to as "dileucine-based," fits the consensus motif (D/E)XXXL(L/I). Previous biochemical analyses showed that (D/E)XXXL(L/I) signals bind to a combination of two subunits of each AP complex, namely the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes, and structural studies revealed that an imperfect variant of this motif lacking the (D/E) residue binds to a site straddling the interface of α and σ2. Herein, we report mutational and binding analyses showing that canonical (D/E)XXXL(L/I) signals bind to this same site on AP-2, and to similar sites on AP-1 and AP-3. The strength and amino acid requirements of different interactions depend on the specific signals and AP complexes involved. We also demonstrate the occurrence of diverse AP-1 heterotetramers by combinatorial assembly of various γ and σ1 subunit isoforms encoded by different genes. These AP-1 variants bind (D/E)XXXL(L/I) signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Our results thus demonstrate that different AP complexes share a conserved binding site for (D/E)XXXL(L/I) signals. However, the characteristics of the binding site on each complex vary, providing for the specific recognition of a diverse repertoire of (D/E)XXXL(L/I) signals.     

Cloning and expression of a plant homologue of the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca acuminata

Clathrin-coated vesicles (CCVs) are involved in selective protein transport in eukaryotes. AP-1 and AP-2 are protein complexes found in the CCVs of the Golgi apparatus and the plasma membrane respectively. AP19 is the smallest polypeptide chain components of AP-1. We have identified a cDNA clone (CAP19) encoding a putative homologue for the assembly protein AP19 from the Chinese medicinal tree, Camptotheca acuminata. The deduced polypeptide contains 161 amino acids and has a predicted M _r of 18 820. DNA blot analysis suggests that the AP19s of C. acuminata are encoded by a small gene family. CAP19 was expressed ubiquitously throughout the plant suggesting that it may be involved in general Golgi-mediated secretion.     

Adaptor complex-independent clathrin function in yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.