Esteban C. Dell'Angelica

AP-3: an adaptor-like protein complex with ubiquitous expression

We have identified two closely related human proteins (σ3A and σ3B) that are homologous to the small chains, σ1 and σ2, of clathrin‐associated adaptor complexes. Northern and Western blot analyses demonstrate that the products of both the σ3A and σ3B genes are expressed in a wide variety of tissues and cell lines. σ3A and σ3B are components of a large complex, named AP‐3, that also contains proteins of apparent molecular masses of 47, 140 and 160 kDa. In non‐neuronal cells, the 47 kDa protein most likely corresponds to the medium chain homolog p47A, and the 140 kDa protein is a homolog of the neuron‐specific protein β‐NAP. Like other members of the medium‐chain family, the p47A chain is capable of interacting with the tyrosine‐based sorting signal YQRL from TGN38. Immunofluorescence microscopy analyses show that the σ3‐containing complex is present both in the area of the TGN and in peripheral structures, some of which contain the transferrin receptor. These results suggest that the σ3 chains are components of a novel, ubiquitous adaptor‐like complex involved in the recognition of tyrosine‐based sorting signals.

Ggas: A Family of Adp Ribosylation Factor-Binding Proteins Related to Adaptors and Associated with the Golgi Complex

Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of γ-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN.

AP-4, a Novel Protein Complex Related to Clathrin Adaptors

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor β and ς subunits named β4 and ς4, respectively. An antibody to β4 recognized in human cells an ∼83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that β4 is a component of a multisubunit complex (AP-4) that also contains the ς4 polypeptide and two additional adaptor subunit homologs named μ4 (μ-ARP2) and ε. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.

β3A-adaptin, a Subunit of the Adaptor-like Complex AP-3

Recent studies have described a widely expressed adaptor-like complex, named AP-3, which is likely involved in protein sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of the subunits of this complex, which we call β3A-adaptin. The predicted amino acid sequence of β3A-adaptin reveals that the protein is closely related to the neuron-specific protein β-NAP (61% overall identity) and more distantly related to the β1- and β2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that β3A-adaptin has a domain organization similar to β-NAP and to β1- and β2-adaptins. β3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that β3A-adaptin corresponds to the ∼140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being δ-adaptin, p47A (now called μ3A) and ς3 (A or B). β3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. β3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of β3A-adaptin reported here lend support to the idea that AP-3 is a structural and functional homolog of the clathrin-associated adaptors AP-1 and AP-2.

Molecular Characterization of the Protein Encoded by the Hermansky-Pudlak Syndrome Type 1 Gene

Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of ∼80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was ∼6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the β3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex.

Purification and partial characterization of a fatty acidbinding protein from the yeast, Yarrowia lipolytica

A low‐molecular‐mass fatty acid‐binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica. Purification was achieved by a two‐step procedure involving size‐exclusion and cation‐exchange chromatography. The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N‐terminus. Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid‐binding proteins.

UNIT 10.16 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.

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.