Juan S. Bonifacino, PhD, Head, Section on Intracellular Protein Trafficking
Rafael Mattera, PhD, Staff Scientist
Patricia Burgos, PhD, Postdoctoral Fellow
Katy Janvier, PhD, Postdoctoral Fellow
Satoshi Kametaka, PhD, Postdoctoral Fellow
Wolf Lindwasser, PhD, Postdoctoral Fellow
Gonzalo Mardones, PhD, Postdoctoral Fellow
Raul Rojas, PhD, Postdoctoral Fellow
William Smith, PhD, Postdoctoral Fellow
Hadiya Watson, PhD, Postdoctoral Fellow
Rittik Chaudhuri, BS, Postbaccalaureate Fellow
Namita Murthy, BS, Postbaccalaureate Fellow
Xiaolin Zhu, RN, Technician

We investigate the molecular mechanisms that determine the sorting of transmembrane proteins to intracellular compartments such as endosomes, lysosomes, and a set of cell type-specific organelles known as lysosome-related organelles. Sorting to these compartments is mediated by signals in the cytosolic domains of the transmembrane proteins, which are recognized by adaptor proteins (APs). Among these APs are the heterotetrameric AP1, AP2, AP3, and AP4 complexes and the monomeric GGA1, GGA2, and GGA3 proteins (see Figure 2.1). Current work in our laboratory aims at elucidating the structure, regulation, and physiological roles of the AP complexes and GGA proteins and investigating the possibility that defects in these proteins underlie certain human diseases.

Missorting of tyrosinase underlies the pigmentation defect in Hermansky-Pudlak syndrome type 2

Martina ¹ ; in collaboration with Marks, Raposo, Theos

The characterization of the molecular machinery involved in protein sorting is important for understanding the pathogenesis of various metabolic and developmental disorders. Several years ago, we discovered, in collaboration with William Gahl (NHGRI), that mutations in the beta3A subunit of AP3 are the cause of the autosomal recessive disorder Hermansky-Pudlak syndrome (HPS) type 2 (HPS2). The disease presents with reduced pigmentation and prolonged bleeding, which are attributable to, respectively, abnormal melanosomes in melanocytes and the absence of dense bodies from platelets. HPS2 is thus a disorder of lysosome-related organelles that affects certain specialized cell types. The connection between the primary AP3 deficiency and the ultimate cellular and organismal phenotypes, however, was not well understood. In collaboration with Alex Theos, Michael Marks, and Graça Raposo, we observed that AP3 is associated with endosomes that are the precursors of melanosomes in melanocytes. In addition, we found that AP3 deficiency prevents transport of tyrosinase, a key enzyme in the biosynthesis of the melanine pigment, from endosomes to melanosomes. Finally, we observed that AP3 binds to a "dileucine-based" signal (EKQPLL in humans; ERQPLL in mice) that is present in the cytosolic tail of tyrosinase. These findings indicate that AP3 is normally involved in the signal-mediated sorting of tyrosinase from endosomes to melanosomes and that AP3 deficiency impairs such sorting, leading to the pigmentation defect that is characteristic of HPS2.

Pleiomorphic carriers mediate cargo transport from the trans-Golgi network to peripheral endosomes

Polishchuk

Recognition of sorting signals by AP1 and GGAs leads to the incorporation of the signal-bearing cargo proteins into transport vesicles that bud from the trans-Golgi network (TGN) and subsequently fuse with endosomes. For a long time, these transport vesicles were thought to be uniformly spherical and to have a diameter of 60-100 nm, but recent fluorescent imaging of live cells showed that they actually have varying shapes and sizes (that is, they are pleiomorphic). Analysis of the ultrastructure of these vesicles with correlative light-electron microscopy (CLEM) revealed that they range from typical 60-100 nm coated vesicles to larger, convoluted tubular-vesicular structures that contain several coated buds. After detaching from the TGN, some of these pleiomorphic structures move long distances in the cytoplasm until they eventually fuse with peripheral endosomes. We propose that pleiomorphic vesicles serve as vehicles for long-range distribution of biosynthetic or recycling cargo from the TGN to peripheral endosomes. The vesicles represent a novel type of coated vesicular intermediate involved in selective cargo transport between membrane-bound compartments.

Accessory proteins that mediate protein transport from the trans-Golgi network: studies of the Rabaptin-5-Rabex-5 complex

Mattera; in collaboration with Hurley, Lee, Weissman

The AP complexes and GGAs have "ear-like" domains that bind to accessory proteins. Most accessory proteins are thought to regulate the budding of transport vesicles and incorporation of cargo proteins into these vesicles. However, work in our laboratory suggests that at least one of these accessory proteins, the Rabaptin-5-Rabex-5 complex, regulates vesicle fusion of TGN-derived transport vesicles with endosomes. We previously showed that the Rabaptin-5-Rabex-5 complex regulates such fusion by binding to the ear domains of both the AP1 gamma subunit and the GGAs via a canonical motif shared with other accessory proteins. Over the past year, we found that ubiquitin regulates the Rabaptin-5-Rabex-5 complex. Mutational and structural analyses (the latter in collaboration with James Hurley) showed that Rabex-5 has two binding sites for ubiquitin. The first site is a zinc finger (ZnF) that binds to a polar region centered on aspartate-58 of ubiquitin, whereas the second site is a new type of ubiquitin-binding domain, an alpha-helical, inverted ubiquitin-interacting motif (IUIM) that binds to a hydrophobic patch centered on isoleucine-44 of ubiquitin. This bipartite binding represents a novel mechanism of ubiquitin recognition that allows the formation of higher-order Rabex-5-ubiquitin structures. Mutation of ubiquitin-binding residues in Rabex-5 impairs its recruitment to endosomes and its ability to mediate endosome fusion, thus demonstrating an important role for ubiquitin in regulating the fate of transport vesicles.

Role of sorting nexins 1 and 2 in the trafficking of mannose 6-phosphate receptors

Rojas, Kametaka; in collaboration with Haft, Hurley, Shi

Newly made lysosomal hydrolases are sorted by binding to mannose 6-phosphate receptors (MPRs) at the TGN. The hydrolase-receptor complexes are recognized by the GGAs, 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 Vps29 and Vps35, which are subunits of a protein complex named retromer, play a role in this retrograde transport of MPRs from endosomes to the TGN. We recently examined the requirement of two other putative subunits of retromer, the sorting nexins 1 and 2 (SNX1 and SNX2). We found that depletion of either SNX protein by RNA interference had no effect on MPR trafficking, but combined depletion of both SNX proteins impaired the recycling of MPRs to the TGN and caused their missorting to lysosomes, where they were degraded. These findings demonstrated that, as part of the retromer complex, SNX1 and SNX2 play interchangeable but essential roles in the sorting of MPRs from endosomes to the TGN.

To elucidate the structural bases for the role of retromer in MPR retrograde transport, we again collaborated with James Hurley to solve the crystal structure of another retromer subunit, Vps26. The structure showed that Vps26 consists of two curved beta-sandwich domains (N and C) that are connected by a polar core and a flexible linker. The structure resembles that of arrestins, which are adaptor molecules involved in the internalization of and signaling by activated G protein-coupled receptors. Arrestins are known to undergo dramatic conformational changes upon interaction with the phosphorylated receptor tails. The changes involve a reorientation of the two beta-sandwich domains such that they embrace the receptor tails through their concave surfaces. The resemblance to the arrestins suggests that Vps26 could undergo a similar conformational change in the process of recognizing the cytosolic tail of some transmembrane protein. Thus, Vps26 could be a cargo-recognition component of the retromer complex.

¹ José Martina, PhD, former Postdoctoral Fellow

COLLABORATORS

Carol Haft, PhD, Division of Diabetes, Endocrinology and Metabolic Diseases, NIDDK, Bethesda, MD
James Hurley, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Sangho Lee, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Michael Marks, PhD, University of Pennsylvania Medical School, Philadelphia, PA
Roman Polishchuk, PhD, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy
Graça Raposo, PhD, Institut Curie, Paris, France
Hang Shi, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Alex Theos, PhD, University of Pennsylvania Medical School, Philadelphia, PA
Allan Weissman, MD, Laboratory of Protein Dynamics and Signaling, NCI, Frederick, MD

For further information, contact bonifacinoj@mail.nih.gov.