PROTEIN SORTING IN THE ENDOSOMAL-LYSOSOMAL SYSTEM
, Head, Section on Intracellular Protein Trafficking
Rafael Mattera, PhD, Staff Scientist
Patricia Burgos, PhD, Postdoctoral Fellow
Luis da Silva, PhD, Postdoctoral Fellow
Katy Janvier, PhD, Postdoctoral Fellow
Satoshi Kametaka, PhD, Postdoctoral Fellow
Wolf Lindwasser, PhD, Postdoctoral Fellow
Gonzalo Mardones, PhD, Postdoctoral Fellow
Javier Pérez-Victoria, PhD, Postdoctoral Fellow
Yogikala Prabhu, PhD, Postdoctoral Fellow
Raúl Rojas, PhD, Postdoctoral Fellow
William Smith, PhD, Postdoctoral Fellow
Hadiya Watson, PhD, Postdoctoral Fellow
Rittik Chaudhuri, BS, Postbaccalaureate Fellow
Namita Murthy, BS, Postbaccalaureate Fellow
Peter Yang, BS, Postbaccalaureate Fellow
Xiaolin Zhu, RN, Technician
Figure 2.1 Structure of AP complexes and GGAs
Regulation of adaptor function by phosphoinositides and accessory proteins
In previous work, we defined many of the structural and functional characteristics of the AP complexes and GGA proteins. Most recently, we made significant progress in understanding the factors that regulate AP and GGA protein function. The association of these adaptors with specific membranes is in part dictated by their ability to bind to certain membrane lipids. In collaboration with Helen Yin, we found that the GGAs bind to phosphatidylinositol 4-phosphate (PI4P), a phosphoinositide lipid that is highly enriched in the trans-Golgi network (TGN). In addition to specifying recruitment to the TGN, binding to PI4P promotes the recognition of ubiquitinated proteins. These observations explain how the GGAs participate in the sorting of ubiquitinated transmembrane cargo at the TGN.
The AP complexes and GGAs have “ear” domains that bind to accessory proteins. We previously demonstrated that these binding interactions are mediated by a canonical sequence motif shared by the accessory proteins. The physiological relevance of the interactions, however, had not been established. This past year, we examined the significance of interactions of the ear domains of the AP and GGA proteins with two accessory proteins, GAK and p56. We found that depletion of both GAK and p56 by RNA interference (RNAi) resulted in missorting of mannose 6-phosphate receptors (MPRs) and their cargo lysosomal hydrolases, resulting in a phenotype similar to that of certain lysosomal storage disorders. The defect could be reversed by transfection with the wild-type GAK or p56 but not with mutant GAK or p56 bearing substitutions in either protein’s ear-binding motifs. Thus, we demonstrated for the first time that the ability of AP complexes and GGAs to engage in canonical ear-motif interactions is critical for protein sorting and lysosome biogenesis.
Kametaka S, Moriyama K, Burgos PV, Eisenberg E, Greene LE, Mattera R, Bonifacino JS. Canonical interaction of cyclin G associated kinase with adaptor protein 1 regulates lysosomal enzyme sorting. Mol Biol Cell 2007;18:2991-3001.
Mardones GA, Burgos PV, Brooks DA, Parkinson-Lawrence E, Mattera R, Bonifacino JS. The trans-Golgi network accessory protein p56 promotes long-range movement of GGA/clathrin-containing transport carriers and lysosomal enzyme sorting. Mol Biol Cell 2007;18:3486-501.
Wang J, Sun HQ, Macia E, Kirchhausen T, Watson H, Bonifacino JS, Yin HL. PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal. Mol Biol Cell 2007;18:2646-55.
Participation of the AP2 complex in the mechanism of CD4 downregulation by the Nef protein of immunodeficiency viruses
Intracellular pathogens manipulate the protein transport machinery of their host cells in order to establish a successful infection. Immunodeficiency viruses, for example, encode an accessory protein, Nef, which downregulates the CD4 co-receptor from the surface of T cells and macrophages. This downregulation is pivotal to the progression from infection to acquired immunodeficiency syndrome (AIDS), making Nef a critical effector of viral pathogenesis. While inhibition of Nef effects could have therapeutic applications, the mechanism by which Nef causes CD4 downregulation is not well understood. We performed an RNAi screen of over 70 components of the protein trafficking machinery to identify host cell factors that are required for Nef function. The screen led to the identification of clathrin and AP2, but not of AP1, AP3, the GGAs, or retromer, as critical mediators of CD4 downregulation by Nef. Given that AP2 is a component of plasma membrane clathrin coats, our observations indicate that Nef enhances the rate of endocytosis of CD4 from the cell surface. We also demonstrated that Nef directly interacts with AP2, with the interaction mediated by both a dileucine-based motif and a novel diacidic-based motif in Nef. In addition, we found that the binding sites for the motifs are located on the alpha and sigma2 subunits of AP2. We discovered that both motifs are required for CD4 downregulation by Nef, thus demonstrating the functional significance of the interactions. The elucidation of this molecular mechanism presents new opportunities for the development of pharmacological agents to inhibit Nef action.
Chaudhuri R, Lindwasser OW, Smith WJ, Hurley JH, Bonifacino JS. Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. J Virol 2007;81:3877-90.
Lindwasser OW, Chaudhuri R, Bonifacino JS. Mechanisms of CD4 downregulation by the Nef and Vpu proteins of primate immunodeficiency viruses. Curr Mol Med 2007;7:171-84.
Role of the retromer complex in retrograde transport from endosomes to the trans-Golgi network
Newly made acid hydrolases are sorted by binding to MPRs at the TGN. The hydrolase-receptor complexes are recognized by the GGA proteins, which mediate packaging into transport vesicles bound for endosomes. The acidic environment of endosomes induces the release of the hydrolases from the MPRs, after which the hydrolases follow the fluid phase to lysosomes while the MPRs return to the TGN to mediate further rounds of transport. In previous work, we showed that the proteins Vps26, Vps29, and Vps35, which are subunits of a protein complex named the retromer, play a role in this retrograde transport of MPRs from endosomes to the TGN. We recently examined the requirement for two other putative subunits of the retromer, the sorting nexins 1 and 2 (SNX1 and SNX2). Using RNA interference, we found that depletion of either SNX protein by RNAi had no effect on MPR trafficking but that combined depletion of both SNX proteins impaired the recycling of the MPR to the TGN and caused its missorting to lysosomes, where it is degraded. As a consequence, lysosomal enzymes were missorted into the extracellular space, a phenotype that is typical of lysosomal storage disorders. Our findings demonstrate that, as part of the retromer complex, SNX1 and SNX2 play interchangeable but essential roles in sorting MPRs from endosomes to the TGN.
To elucidate the structural bases for the role of retromer in MPR retrograde transport, we collaborated with James Hurley, Alasdair Steven, and their colleagues to solve the structure of the retromer Vps26–Vps29–Vps35 subcomplex by X-ray crystallography and electron microscopy. We found that the complex is a rod of about 21nm with Vps26 at one end and Vps29 at the other. Vps26 is structurally similar to arrestins, whereas Vps29 resembles a type of metallophophoesterase. Vps35 consists of a long alpha-helical solenoid that spans the length of the rod and covers the putative metallophosphoesterase active site on Vps29. Vps35 also exposes several grooves that could be binding sites for sorting signals on cargo molecules such as MPRs.
The retromer complex is not only used to retrieve intracellular sorting receptors from endosomes to the TGN but is also exploited by certain bacterial toxins to access their target compartments. An example is Shiga toxin, which we have shown, in collaboration with Ludger Johannes and Graça Raposo, requires retromer for its movement from endosomes to the TGN. Transport begins at vacuolar, early endosomes and proceeds through tubules that are part of what we refer to as the tubular endosomal network.
Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol 2006;7:568-79.
Hierro A, Rojas AL, Rojas R, Murthy N, Effantin G, Kajava AV, Steven AC, Bonifacino JS, Hurley JH. Functional architecture of the retromer cargo-recognition complex. Nature 2007;449:4063-7.
Popoff V, Mardones GA, Tenza D, Rojas R, Lamaze C, Bonifacino JS, Raposo G, Johannes L. The retromer complex and clathrin define an early endosomal retrograde exit site. J Cell Sci 2007;120:2022-31.
Rojas R, Kametaka S, Haft CR, Bonifacino JS. Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol Cell Biol 2007;27:1112-24.
Protein sorting to the multivesicular body pathway
The sorting of integral membrane proteins into the lumen of lysosomes involves passage through an intermediate organelle known as the multivesicular body (MVB). For most proteins, the sorting involves recognition of ubiquitinated proteins by several complexes known as ESCRT. In collaboration with James Hurley and colleagues, we solved the crystal structure of the ESCRT Vps27–Hse1 complex. The core of the complex is formed by two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The domains are similar to the previously described GAT domain of the GGAs. The two Vps27-Hse1 GAT domains are connected by an antiparallel coiled-coil to form a 90 Å–long barbell-like structure. Our studies explain how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.
While most proteins require ubiquitination for targeting to the MVB pathway, the yeast transmembrane protein Sna3p is an exception. Despite the fact that Sna3p itself is not ubiquitinated, we have found that the ubiquitin ligase Rsp5 and the ESCRT complexes are nonetheless required for Sna3p targeting to the MVB pathway. Such targeting is mediated by a direct interaction between a PPAY (proline-proline-alanine-tyrosine) motif within the Sna3p C-terminal cytosolic domain and the WW domains of Rsp5p. Sna3p is thus an example of a new class of proteins that follow a ubiquitination-independent, but ubiquitin-ligase–mediated, sorting pathway to the vacuole.
Prag G, Watson H, Kim YC, Beach BM, Ghirlando R, Hummer G, Bonifacino JS, Hurley JH. The Vps27/Hse1 complex is a GAT domain-based scaffold for ubiquitin-dependent sorting. Dev Cell 2007;12:973-86.
Watson H, Bonifacino JS. Direct binding to Rsp5p regulates ubiquitination-independent vacuolar transport of Sna3p. Mol Biol Cell 2007;18:1781-9.
1 Kengo Moriyama, PhD, former Postdoctoral Fellow
COLLABORATORS
Doug Brooks, PhD, University of South Australia, Adelaide, Australia
Evan Eisenberg, PhD, Laboratory of Cell Biology, NHLBI, Bethesda, MD
Lois Greene, PhD, Laboratory of Cell Biology, NHLBI, Bethesda, MD
Carol Haft, PhD, Division of Diabetes, Endocrinology and Metabolic Diseases, NIDDK, Bethesda, MD
Aitor Hierro, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
James Hurley, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Ludger Johannes, PhD, Institut Curie, Paris, France
Gali Prag, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Graça Raposo, PhD, Institut Curie, Paris, France
Adriana Rojas, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Alasdair C. Steven, PhD, Laboratory of Structural Biology, NIAMS, Bethesda, MD
Helen Yin, PhD, University of Texas Southwestern Medical Center, Dallas, TX
For more information, contact bonifacinoj@mail.nih.gov.
