We investigate the molecular mechanisms that control the sorting of transmembrane proteins in the endosomal-lysosomal system. Sorting processes such as rapid internalization of receptors from the plasma membrane, targeting to lysosomes and related organelles, and delivery to the basolateral plasma membrane domain of polarized epithelial cells are all mediated by interactions between signals in the cytosolic domains of the transmembrane proteins and adaptor proteins associated with the cytosolic face of membranes. Two major types of sorting signal, referred to as tyrosine-based and dileucine-based, have been previously described. Work in our as well as in other laboratories has demonstrated that both types of signal are recognized with characteristic fine specificities by the adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 or the GGA proteins GGA1, GGA2, and GGA3 (Fig. 1). Mutations in AP-3 are the cause of the pigmentation and bleeding disorder Hermansky-Pudlak syndrome type 2. Current work is aimed at elucidating the structure, regulation, and physiological roles of AP complexes and GGAs and at investigating the possibility that defects in these proteins underlie protein trafficking disorders.

Role of the GGAs in the Sorting of Lysosomal Hydrolases


Puertollano, Bonifacino
Over the past year, we continued our studies on the structure and function of the GGAs. The GGAs are modular proteins composed of four domains named the VHS, GAT, hinge, and GAE domains. Previous work in our laboratory demonstrated that the GGAs function as ARF-dependent adaptors for recruitment of clathrin to the TGN. We discovered that the VHS domain of the GGAs functions as a recognition module for dileucine-based sorting signals present in the cytosolic domains of the mannose 6-phosphate receptors (MPRs) that sort acidic hydrolases to lysosomes. In collaboration with James Hurley’s group (NIDDK), we solved the crystal structure of a GGA VHS domain in complex with signals from the MPRs. The structure revealed the basis for the specificity of recognition of these signals. In addition, we demonstrated that casein kinase II–mediated phosphorylation of a serine residue within the MPR dileucine–based signal enhances interactions with the GGA VHS domain, thus explaining the regulation of this sorting process by phosphorylation. The studies have therefore solved the longstanding enigma of how the MPRs are able to sort acidic hydrolases to lysosomes. The knowledge gained from the biochemical and structural characterization of these interactions allowed us to predict a role for the GGAs in the sorting of other physiologically important proteins such as beta-secretase, the enzyme responsible for the generation of the beta-amyloid precursor associated with Alzheimer’s disease.

A Novel Type of Coated Vesicle Budding from the Trans-Golgi Network
Puertollano, Bonifacino
Sorting of MPRs and their hydrolase ligands at the trans-Golgi network (TGN) has long been thought to be mediated by small (60–100 nm diameter) clathrin-coated vesicles. We have conducted experiments using fluorescent imaging of live cells that suggest a role for a different type of clathrin-coated intermediate. Clathrin and GGA1, labeled with different spectral variants of the green fluorescent protein (GFP), colocalize to the TGN and to a population of vesicles budding from it. Strikingly, these vesicles are pleiomorphic and have average diameters of 360 to 380 nanometers. They move toward peripheral cytoplasm for distances of up to 10 micrometers with average speeds of about one micrometer per second. Independently of vesicle budding, the labeled clathrin and GGA1 cycle on and off membranes with half-times of 10 to 20 seconds. These observations suggest the existence of a novel type of clathrin-coated carrier that is both larger than conventional clathrin-coated vesicles and undergoes long-range translocation in the cytoplasm before losing its coat. The carriers are likely the intermediates that ferry acidic hydrolases from the TGN to the endosomal-lysosomal system.

Interaction of TGN-Derived Carriers with Endosomes
Mattera, Puertollano, Bonifacino
The transfer of cargo from the clathrin- and GGA1-coated carriers to endosomes involves fusion or “kiss-and-run” interactions between these two types of organelle. We have identified a molecular interaction that may be responsible for the transfer. The GGAs have been found to bind via their GAE domains to the Rabaptin-5/Rabex-5 complex. The complex functions as a guanine nucleotide exchange factor for Rab5 and Rab4 and has been previously implicated in endosomal fusion reactions. The GAE domain binds to an FGPLV motif in the central region of Rabaptin-5.

Biogenesis of Lysosome-Related Organelles
Martina, Moriyama, Bonifacino
We have previously shown that mutations in the gene encoding the beta3A subunit of AP-3 are the cause of Hermansky-Pudlak syndrome type 2, a disorder of lysosome-related organelles. Mutations in at least three other genes in humans and 14 genes in mice have been found to cause a similar disorder. Most of the HPS genes identified so far by positional cloning encode proteins of unknown function and no recognizable homology to other proteins. To gain insight into the nature of this machinery, we have undertaken a biochemical characterization of the novel HPS gene products. We have found that the protein products of the pallid, muted, and cappuccino genes are the components of a novel complex. The complex has two additional subunits of approximately 20kD and 15kD, which we are aiming to identify by affinity purification and mass spectrometry. Current studies are directed toward determining the intracellular localization and function of the complex. From these experiments, we hope to gain an understanding of the molecular mechanisms involved in the biogenesis of lysosome-related organelles and the pathogenesis of Hermansky-Pudlak syndrome.

A Genomic Screen for Vacuolar Protein Sorting Genes in Yeast
Bonangelino, Chavez, Bonifacino
The yeast vacuole is the functional counterpart of mammalian lysosomes. Thus, yeast is an excellent model system for investigating the basic molecular mechanisms involved in lysosome biogenesis. To identify genes involved in vacuolar protein sorting, we conducted a genome-wide screen of 4,653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of the vacuolar hydrolase, carboxypeptidase Y (CPY). We identified 149 mutant strains that secreted strong to moderate levels of CPY. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting while the remaining 96 either had been identified in screens for other cellular processes or were known only as hypothetical open reading frames. Among the 96 are genes encoding (1) the four subunits of the AP-3 complex; (2) the Ras-like GTP-binding proteins, Arl1p and Arl3p; (3) actin-related proteins such as Arp5p and Arp6p; (4) the monensin and brefeldin A hypersensitivity proteins, Mon1p and Mon2p; and (5) 16 novel proteins designated Vps61p–Vps76p. Mutations in several of the novel proteins, including Vps61p, Vps64p, and Vps67p, resulted in actin cytoskeleton defects. The identification and phenotypic characterization of these novel mutants provide powerful new insights into the biogenesis of the yeast vacuole and mammalian lysosomes, most notably the involvement of the actin cytoskeleton in this process.

A Tubular Intermediate in Protein Recycling to the Plasma Membrane
Caplan, Bonifacino
Pivotal to the function of many plasma membrane proteins is their ability to undergo endocytosis and recycling to the cell surface. We have uncovered a role for a protein named EHD1 in the recycling of plasma membrane proteins such as class I molecules of the major histocompatibility complex to the cell surface. EHD1 cooperates with the small GTP-binding protein ARF6 to induce the formation of tubules containing internalized proteins that extend from the center toward the periphery of the cells. The discovery of the role of EHD1 in this process opens the way for the identification of other components of the molecular machinery involved in recycling to the plasma membrane.