We investigate the molecular mechanisms that control the sorting of transmembrane proteins in the endosomal-lysosomal system. Sorting events such as rapid internalization from the plasma membrane, transport to lysosomes and lysosome-related organelles, and delivery to the basolateral plasma membrane domain of polarized epithelial cells are all mediated by recognition of specific signals in the cytosolic domains of the transmembrane proteins by adaptor proteins associated with the cytosolic face of membranes. Among these adaptors are the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 and the monomeric GGA1, GGA2, and GGA3 proteins (see Figure 2.1). Mutations in AP-3 are the cause of the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) type 2. Current work focuses on elucidating the structure, regulation, and physiological roles of the AP complexes and GGA proteins and other adaptors as well as on the possibility that defects in or interference with these proteins underlie certain human diseases.
Role of GGA proteins in sorting to lysosomes
Mattera, Kametaka; in collaboration with Hurley, Prag
Over the past year, we have continued our studies on the structure and function of GGAs, proteins that are composed of four domains named VHS, GAT, hinge, and GAE (see Figure 2.1). The VHS domain is a recognition module for a subset of “dileucine-based” sorting signals present in the cytosolic domains of the mannose 6-phosphate receptors (MPRs) that sort acidic hydrolases to lysosomes. The GAT domain binds to GTP-Arf and targets GGAs to the trans-Golgi network (TGN) and endosomes. In addition, the GAT domain binds to ubiquitin, Rabaptin-5, and the tumor susceptibility gene-101 product (TSG101). The hinge domain recruits clathrin. Finally, the GAE domain binds to accessory factors involved in vesicle budding and fusion and in interactions with the cytoskeleton. These properties indicate that GGAs function as Arf-dependent adaptors for the recruitment of clathrin to membranes and for sorting of MPRs and ubiquitinated transmembrane proteins at the TGN and endosomes. This past year, we collaborated with James Hurley and colleagues to elucidate the structural basis for the interaction of the GAT domain of GGA3 with ubiquitin. The GAT domain consists of two subdomains, a “hook” that binds to GTP-Arf and a “trihelical bundle” that binds to ubiquitin. Ubiquitin was found to bind to a hydrophobic patch made up of residues on helices 1 and 2 of the bundle (see Figure 2.2). The site does not overlap with the binding sites for Rabaptin-5 and TSG101. The GAT domain therefore functions as a hub for interactions with several components of the protein-trafficking machinery. The ability of the GAT domain to bind to ubiquitin is thought to be important for the role of GGAs in targeting transporters such as the general amino acid permease Gap1p and the internalized epidermal growth factor (EGF) receptor for degradation in lysosomes.
The involvement of GGAs in many interactions and sorting events likely requires precise regulatory controls. This past year, we found that phosphorylation plays a role in the regulation of GGA function. Activation of the EGF receptor leads to phosphorylation of GGA3 on serine-368, a hinge residue that is strictly dependent on the constitutive phosphorylation of serine-372, another hinge residue. The EGF-induced phosphorylation causes a conformational change in GGA3, as evidenced by an increase in its hydrodynamic radius. In addition, the phosphorylation decreases GGA3’s association with membranes and its affinity for ubiquitin. These observations indicate that EGF signaling elicits phosphorylation events that regulate the association of GGA3 with organellar membranes. Such regulation could be responsible for the reported secretion of lysosomal hydrolases upon treatment of cells with growth factors or in oncogenically transformed cells.
Kametaka S, Mattera R, Bonifacino JS. Epidermal growth factor-dependent phosphorylation of the GGA3 adaptor protein regulates its recruitment to membranes. Mol Cell Biol2005;25:7988-8000.
Prag G, Lee S, Mattera R, Arighi CN, Beach BM, Bonifacino JS, Hurley JH. Structural mechanism for ubiquitinated cargo recognition by the GGA proteins. Proc Natl Acad Sci USA 2005;102:2334-2339.
Role of AP complexes in protein trafficking
Janvier, McCormick, Martina1; in collaboration with Ohno, Rose, Venkatesan
In addition to GGAs, the heterotetrameric AP complexes AP-1, AP-2, AP-3, and AP-4 play important roles in protein trafficking. The complexes recognize tyrosine-based signals as well as a subset of dileucine-based sorting signals that are distinct from those recognized by GGAs. The limiting membrane of the lysosome is enriched in a group of transmembrane glycoproteins, named lysosome-associated membrane proteins (LAMPs), that contain either type of signal. However, the roles of the four AP complexes and the coat protein clathrin in sorting the LAMPs in vivo had not been assessed or was controversial before we initiated our work. In addition, it was unclear whether LAMPs are transported from the TGN directly to endosomes and lysosomes or indirectly via the plasma membrane. We used RNA interference (RNAi) to show that AP-2 and clathrin (and to a lesser extent the other AP complexes) are required for efficient delivery of LAMPs to lysosomes. Given that AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of LAMPs traffic via the plasma membrane en route to lysosomes.
Major histocompatibility complex class II (MHC-II) molecules are also sorted to the endosomal-lysosomal system by virtue of dileucine-based sorting signals. The molecules are composed of two polymorphic chains, alpha and beta, that assemble with Ii, an invariant chain, in the endoplasmic reticulum. The assembled MHC-II complexes are transported to the Golgi complex and then to late endosomes and lysosomes, where Ii is degraded and alpha-beta dimers bind to peptides derived from exogenous antigens. Targeting of MHC-II molecules to these compartments is mediated by two dileucine-based signals in the cytoplasmic domain of Ii. While the signals bind in vitro to AP-1 and AP-2, the physiological roles of the proteins in MHC-II trafficking in vivo have remained poorly understood. The use of RNAi allowed us to determine that depletion of clathrin or AP-2 caused a greater than 10-fold increase in Ii expression on the cell surface and a concomitant decrease in Ii localization to endosomal/lysosomal vesicles. In addition, depletion of clathrin or AP-2 delayed the degradation of Ii and reduced the surface expression of peptide-loaded alpha-beta dimers. In contrast, depletion of AP-1, AP-3, or AP-4 had little or no effect. The findings demonstrated that clathrin and AP-2 participate in MHC-II molecule trafficking in vivo. As is the case for the Ls, the results indicated that a significant pool of MHC-II molecules traffic to the endosomal-lysosomal system by means of the cell surface.
We collaborated with Sundararajan Venkatesan and colleagues to investigate the role of AP complexes in the downregulation of the T-lymphocyte co-receptor CD4 by the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV, respectively). It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 complex. However, the related AP-1 and AP-3 adaptors also interact with Nef, hinting at roles for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA–induced knockdown of AP-2, we found that accelerated CD4 endocytosis is not a dominant mechanism for HIV-1 Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we observed that both CD4 recycling from the plasma membrane and nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef–expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV Nef–induced downregulation of CD4 in human cells. Our observations shed new light on the mechanisms by which HIV and SIV interfere with their host cells.
The observations described above indicated that the AP-2 complex plays important roles not only in receptor endocytosis but also in the targeting of proteins to various compartments of the endosomal/lysosomal system. To determine whether this function is essential in the context of a whole organism, we carried out, in collaboration with Hiroshi Ohno and colleagues, targeted disruption of the gene encoding the mu2 subunit of AP-2 in mouse. We found that mu2 heterozygous mutant mice were viable and had an apparently normal phenotype. By contrast, we identified no mu2 homozygous mutant embryos among blastocysts from intercrossed heterozygotes, demonstrating that mu2-deficient embryos die before day 3.5 post-coitus. The results indicate that AP-2 is indispensable for early embryonic development, perhaps because it is a requirement for cell viability.
Janvier K, Bonifacino JS. Role of the endocytic machinery in the sorting of lysosome-associated membrane proteins. Mol Biol Cell 2005; 16:4231-4242.
McCormick PJ, Martina JA, Bonifacino JS. Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments. Proc Natl Acad Sci USA2005;102:7910-7915.
Mitsunari T, Nakatsu F, Shioda N, Love PE, Grinberg A, Bonifacino JS, Ohno H. Clathrin adaptor AP-2 is essential for early embryonal development. Mol Cell Biol 2005;25:9318-9323.
Rose JJ, Janvier K, Chandrasekhar S, Sekaly RP, Bonifacino JS, Venkatesan S. CD4 downregulation by HIV-1 and SIV Nef proteins involves both internalization and intracellular retention mechanisms. J Biol Chem 2005;280:7413-7426.
The biogenesis of lysosome-related organelles
Lefrançois, Janvier, Martina, 1 Moriyama2; in collaboration with Ciciotte, Gwynn, Marks, Peters, Raposo
The characterization of the molecular machinery involved in protein sorting is important for understanding the pathogenesis of various metabolic and developmental disorders. An example is Hermansky-Pudlak syndrome (HPS), a genetically heterogeneous disease that affects lysosome-related organelles such as melanosomes and platelet dense bodies. In past studies, we discovered that mutations in the gene encoding the beta3A subunit of AP-3 are the cause of HPS type 2. This past year, we uncovered an intramolecular interaction that regulates the recruitment of AP-3 to membranes. We found that the “ear” domain of the delta subunit binds to the sigma3 subunit of the same complex. The interaction blocks binding of AP-3 to GTP-Arf1, thus preventing AP-3 recruitment to membranes. Therefore, we envision that AP-3 exists in a “closed,” inactive conformation in the cytosol, which needs to be activated for recruitment of AP-3 to membrane-bound GTP-Arf1. In addition to mutations in the beta3A gene, mutations in at least six other genes in humans and 14 genes in mice cause an HPS-like disorder. Most of the HPS genes identified to date by positional cloning encode proteins of unknown function and no recognizable homology to other proteins. We and others have shown that some of the proteins are components of three complexes named BLOC-1, BLOC–2, and BLOC-3. This past year, in collaboration with Luanne Peters and colleagues, we identified a new subunit of BLOC-1 that is encoded by the “reduced pigmentation” gene. Finally, in collaborative studies with Graça Raposo and Michael Marks and their respective colleagues, we found that AP-3 and AP-1 function in partially redundant pathways for the transfer of cargo from distinct endosomal subdomains to melanosomes and other lysosome-related organelles. Ongoing studies on the AP and BLOC complexes are likely to provide additional insights into the pathogenesis of HPS.
Gwynn B, Martina JA, Bonifacino JS, Sviderskaya EV, Lamoreux ML, Bennett DC, Kengo K, Huizing M, Helip-Wooley A, Gahl WA, Webb LS, Lambert AJ, Peters LL. Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex. Blood 2004;104:3181-3189.
Lefrançois S, Janvier K, Boehm M, Ooi CE, Bonifacino JS. An ear-core interaction regulates the recruitment of the AP-3 complex to membranes. Dev Cell 2004;7:619-625.
Theos AC, Tenza D, Martina JA, Hurbain I, Peden AA, Sviderskaya E, Stewart A, Robinson MS, Bennett DC, Cutler DF, Bonifacino JS, Marks MS, Raposo G. Tyrosinase sorting to melanosomes reveals a general role for AP-3 and AP-1 in trafficking from endosomes. Mol Biol Cell 2005; 16:5356-5372.
1Jose Martina, PhD, former Postdoctoral Fellow
2Kengo Moriyama, PhD, former Postdoctoral Fellow
COLLABORATORS
Steve Ciciotte, BS, The Jackson Laboratory, Bar Harbor, ME
Babette Gwynn, MS, The Jackson Laboratory, Bar Harbor, ME
James H. Hurley, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Michael Marks, PhD, University of Pennsylvania School of Medicine, Philadelphia, PA
Hiroshi Ohno, MD, Riken, Yokohama, Japan
Luanne Peters, PhD, The Jackson Laboratory, Bar Harbor, ME
Gali Prag, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Graça Raposo, PhD, Curie Institute, Paris, France
Jeremy Rose, MS, Laboratory of Molecular Microbiology, NIAID, Bethesda, MD
Sundararajan Venkatesan, PhD, Laboratory of Molecular Microbiology, NIAID, Bethesda, MD
For further information, contact bonifacinoj@mail.nih.gov.