Structural requirements for function of yeast GGAs in vacuolar protein sorting, alpha-factor maturation, and interactions with clathrin.

The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of alpha-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and alpha-factor maturation and identify structural determinants that are critical for these functions.

Functional and physical interactions of the adaptor protein complex AP-4 with ADP-ribosylation factors (ARFs).

AP-4 is a member of the family of heterotetrameric adaptor protein (AP) complexes that mediate the sorting of integral membrane proteins in post-Golgi compartments. This complex consists of four subunits (epsilon, beta4, mu4 and sigma4) and localizes to the cytoplasmic face of the trans-Golgi network (TGN). Here, we show that the recruitment of endogenous AP-4 to the TGN in vivo is regulated by the small GTP-binding protein ARF1. In addition, we demonstrate a direct interaction of the epsilon and mu4 subunits of AP-4 with ARF1. epsilon binds only to ARF1-GTP and requires residues in the switch I and switch II regions of ARF1. In contrast, mu4 binds equally well to the GTP- and GDP-bound forms of ARF1 and is less dependent on switch I and switch II residues. These observations establish AP-4 as an ARF1 effector and suggest a novel mode of interaction between ARF1 and an AP complex involving both constitutive and regulated interactions.

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-related proteins.

Two new adaptor-related protein complexes, AP-3 and AP-4, have recently been identified, and both have been implicated in protein sorting at the trans-Golgi network (TGN) and/or endosomes. In addition, two families of monomeric proteins with adaptor-related domains, the GGAs and the stoned B family, have also been identified and shown to act at the TGN and plasma membrane, respectively. Together with the two conventional adaptors, AP-1 and AP-2, these proteins may act to direct different types of cargo proteins to different post-Golgi membrane compartments.

Human Vam6p promotes lysosome clustering and fusion in vivo.

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH(2) terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2-3 microm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Sorting of mannose 6-phosphate receptors mediated by the GGAs.

The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.

Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery.

Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Here, we report the identification of two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery. The human stonins are homologous to the Drosophila melanogaster stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery.

The GGAs promote ARF-dependent recruitment of clathrin to the TGN.

The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.

The molecular machinery for lysosome biogenesis.

The lysosome serves as a site for delivery of materials targeted for removal from the eukaryotic cell. The mechanisms underlying the biogenesis of this organelle are currently the subject of renewed interest due to advances in our understanding of the protein sorting machinery. Genetic model systems such as yeast and Drosophila have been instrumental in identifying both protein and lipid components of this machinery. Importantly, many of these components, as well as the processes in which they are involved, are proving conserved in mammals. Other recently identified components, however, appear to be unique to higher eukaryotes. BioEssays 23:333-343, 2001. Published 2001 John Wiley & Sons, Inc.

Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4.

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.

Metabolic labeling with amino acids.

Metabolic labeling techniques are used to study biosynthesis, processing, intracellular transport, secretion, degradation, and physical-chemical properties of proteins. This unit focuses on pulse-labeling and pulse-chase experiments done with [(35)S]-methionine, but gives directions for labeling with other radiolabeled amino acids, and also offers guidance for the safe use and handling of 35S-labeled compounds.

Immunoprecipitation.

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in-vitro-translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear-cut explanation of the options for employing the technology.

Biosynthetic labeling of proteins.

Biosynthetic labeling techniques are commonly used in the study of biochemical properties, synthesis, processing, intracellular transport, secretion, and degradation of proteins. In this unit, protocols are described for biosynthetically labeling many secreted and membrane proteins. The Basic Protocol describes short-term labeling of cells in suspension (30 min to 3 hr) and an alternate protocol describes a modification of this procedure to be used for adherent cells. Alternate protocols for long-term (3 to 24 hr) and pulse-chase labeling of cells are also presented. Label incorporation can be determined as described in a support protocol. The cells used in these protocols can be either from suspension cultures or from single-cell suspensions of spleen or thymus.

Immunoprecipitation.

Immunoprecipitation consists of multiple ordered steps: lysing the cell with detergent if the antigen (usually a protein) to be precipitated is membrane-bound; binding of a specific antigen to an antibody; precipitating the antibody-antigen complex; washing the precipitate; and dissociating the antigen from the immune complex. The dissociated antigen is then analyzed by electrophoretic methods. In this unit, the basic protocol details the immunoprecipitation of a radiolabeled antigen with a specific antibody (polyclonal or monoclonal) covalently linked to Sepharose. Preparation of Ab-Sepharose is described in the Support Protocol. The first two alternate protocols present methods for precipitating or isolating the soluble immune complexes formed between a specific antibody and a radiolabeled antigen. Immunoprecipitation is achieved with polyclonal anti-immunoglobulin (Ig) serum, anti-Ig-Sepharose, Staphylococcus protein A or Streptococcus protein G bound to Sepharose, or Staphylococcus aureus bacteria which contain protein A on the cell surface. The third alternate protocol should be used for immunoprecipitation of antigens that are nonspecifically associated with other proteins. The fourth alternate protocol describes immunoprecipitation of unlabeled protein antigens with Ab-Sepharose.

Staining proteins in gels.

Once proteins are separated by gel electrophoresis, staining can be used to visualize the proteins. This unit presents protocols for numerous staining methods. The most common method is staining with Coomassie blue, which after washing gives blue bands on a clear background. This technique can also be applied to isoelectric focusing gels. A second, more sensitive but also more technically challenging method is silver staining. Here the proteins are seen as dark brown to black bands on a clear background. If the gel is incubated with SYPRO Ruby, a fluorescent compound that interacts specifically with proteins, the bands fluoresce when illuminated on a standard transilluminator. Finally, proteins can be reversibly stained with zinc, which precipitates the SDS from the gel leaving protein bands as clear spots against an opaque white background.

Metabolic labeling with amino acids.

Metabolic labeling techniques are used to study the biosynthesis, processing, intracellular transport, secretion, degradation, and physical-chemical properties of proteins. This unit provides protocols for metabolic labeling of cells with [(35)S]-labeled amino acids to provide material suitable for analysis by immunoprecipitation, for characterization of cellular proteins, for analysis of protein trafficking, and for one- and two-dimensional gel electrophoresis. Three procedures are described for suspension and adherent cells: pulse-labeling, pulse-chase labeling, or continuous long-term labeling. A support protocol describes TCA precipitation to measure the extent of labeling.

Immunoprecipitation.

Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein-protein interactions. Clear step-by-step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody.

Immunoprecipitation.

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in vitro-translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear-cut explanation of the options for employing the technology.

Metabolic labeling with amino acids.

Metabolic labeling techniques are used to study biosynthesis, processing, intracellular transport, secretion, degradation, and physical-chemical properties of proteins. This update focuses on pulse-labeling and pulse-chase experiments done with [(35)S]-methionine, but gives directions for labeling with other radiolabeled amino acids, and also offers guidance for the safe use and handling of 35S-labeled compounds.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3-independent mechanism.

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)