Protein Sorting in the Endosomal-Lysosomal System

Juan S. Bonifacino, PhD, Head, Section on Intracellular Protein Trafficking
Rafael Mattera, PhD, Staff Scientist
Patricia Burgos, PhD, Postdoctoral Fellow
Luis daSilva, PhD, Postdoctoral Fellow
Wolf Lindwasser, PhD, Postdoctoral Fellow
Javier Magadán, PhD, Postdoctoral Fellow
Gonzalo Mardones, PhD, Postdoctoral Fellow
Javier Pérez-Victoria, PhD, Postdoctoral Fellow
Yogikala Prabhu, PhD, Postdoctoral Fellow
Raúl Rojas, PhD, Postdoctoral Fellow
Christina Schindler, PhD, Postdoctoral Fellow
William Smith, PhD, Postdoctoral Fellow
Krishnakant Soni, PhD,Postdoctoral Fellow
Timothy Wagenaar, PhD, Postdoctoral Fellow
Rittik Chaudhuri, BS, Postbaccalaureate Fellow
Namita Murthy, BS, Postbaccalaureate Fellow
Xiaolin Zhu, RN, Technician

We investigate the molecular mechanisms by which transmembrane proteins are sorted to intracellular compartments such as endosomes, lysosomes, and a group of cell-type–specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting to these compartments is mediated by adaptor proteins’ recognition of signals present in the cytosolic domains of the transmembrane proteins; the adaptor proteins are components of membrane coats. Among the adaptor proteins are the heterotetrameric ap-1, ap-2, ap-3, and ap-4 complexes; the monomeric GGA1, GGA2, and GGA3 proteins (GGAs) (Figure 1.1); and the heteropentameric “retromer” complex. Proper sorting requires, in addition to these adaptors, the function of other components of the trafficking machinery that mediate vesicle tethering and fusion. Current work in our laboratory aims to elucidate the structure, regulation, and physiological roles of coat proteins and vesicle tethering factors and to investigate human diseases that result from genetic defects (hermansky-pudlak syndrome) or pathogens’ (HIV-1) exploitation of these proteins.

Figure 1.1 Structure of AP complexes and GGAs

Exploitation of adaptor proteins by HIV-1

Lindwasser, Smith, Chaudhuri, Yang¹; in collaboration with Freed, Guatelli, Hurley

The ap-2 complex comprises four subunits (alpha, beta2, mu2, and sigma2) and is a component of plasma membrane clathrin coats. Its primary function is to mediate clathrin-dependent endocytosis through recognition of endocytic signals in the cytosolic tails of transmembrane proteins. In previous work, we showed that the Nef protein of HIV-1 requires ap-2 for the downregulation of the cd4 co-receptor from the surface of host cells. Formation of a cd4–Nef–ap-2 complex promotes cd4 endocytosis via plasma membrane clathrin-coated pits. In addition, we found that a “dileucine” signal in Nef interacts with a combination of the alpha and sigma2 subunits of ap-2. We have now discovered that a second, “diacidic” motif in Nef interacts with a different binding site on the alpha-sigma2 hemicomplex. Both interactions are required for Nef to downregulate cd4. We are currently conducting further analyses of the tripartite interactions among cd4, Nef, and ap-2 and are examining the mechanism by which internalized cd4 is targeted to lysosomal degradation upon expression of Nef.

Another effect of HIV-1 Nef on host cells is the upregulation of immature class II molecules of the major histocompatibility complex (MHC-II) in association with the invariant chain (Ii). In collaboration with John Guatelli and colleagues, we have found that such upregulation is caused by competition between dileucine signals in Nef and in the cytosolic tail of Ii for binding to the alphasigma2 hemicomplex. Overexpression of Nef during infection saturates the dileucine binding site on AP-2, thus interfering with dileucine- and AP-2–mediated endocytosis of MHC-II-Ii from the cell surface. The trapping of MHC-II-Ii at the cell surface prevents MHC-II from acquiring viral peptides in endosomes, thus interfering with immune surveillance.

In collaboration with Eric Freed and colleagues, we have also examined the role of adaptor proteins in HIV-1 budding from host cells. We found that the GGA proteins, which are monomeric adaptors associated with trans-Golgi network clathrin coats, modulate HIV-1 release into the extracellular space by acting on the small GTP-binding protein Arf.

Chaudhuri R, Lindwasser OW, Smith WJ, Hurley JH, Bonifacino JS. CD4 downregulation by HIV-1 Nef is dependent on clathrin and involves a direct interaction of Nef with the AP2 clathrin adaptor. J Virol 2007;81:3877-3890.
Joshi A, Garg H, Nagashima K, Bonifacino JS, Freed eo. GGA and ARF proteins modulate retrovirus assembly and release. Mol Cell 2008;30:227-238.
Lindwasser OW, Smith WJ, Chaudhuri R, Yang P, Hurley JH, Bonifacino JS. A diacidic motif in HIV-1 Nef is a novel determinant of binding to AP2. J Virol 2008;82:1166-1174.
Mitchell RS, Chaudhuri R, Lindwasser OW, Murillo R, Bonifacino JS, Guatelli JC. Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef. J Virol 2008;82:7758-7767.

Cleft palate caused by a defect in AP-2

Puertollano²; in collaboration with Everett, Overbeek, Gahl

Mutant organisms have previously aided in investigations of the physiological roles of AP complexes. For example, in collaboration with William Gahl (NHGRI), we showed that mutations in one of the subunits of the AP-3 complex in humans cause the pigmentation and bleeding disorder Hermansky- Pudlak syndrome type 2. To investigate the role of AP-2, we previously ablated the gene encoding the mu2 subunit in mice. This approach yielded little information because defective expression of mu2 resulted in early embryonic lethality. In collaboration with Eric Everett and Paul Overbeek, we have now characterized a mutant mouse deficient in expression of the beta2 subunit of AP-2. Unlike the mu2 mutant mouse, the beta2 mutant mouse survives until birth but dies shortly thereafter. The viability of beta2 mutant embryos is attributable to substitution of the beta1 subunit of AP-1 for beta2 in the AP-2 complex. Most interestingly, the newborn beta2 mutant mouse exhibits a single visible developmental defect: cleft palate. Thus, endocytosis must be critical for the development of the palate, probably by promoting the internalization of a key signaling receptor. Our finding identifies a possible cause for cleft palate, which is one of the most frequently occurring congenital deformities in humans.

Structure and regulation of the retromer complex

Rojas, Prabhu, Murthy, Mardones; in collaboration with Hurley, Steven, van der Sluijs

The retromer complex is another sorting device that mediates “retrograde” transport from endosomes to the trans-Golgi network. This function is essential for many important physiological processes in higher eukaryotes, including lysosomal enzyme sorting, processing of the Alzheimer’s disease amyloid precursor protein, and formation of morphogen gradients during development. The retromer comprises a membrane-binding subcomplex made up of two “sorting nexin” (SNX) subunits and a cargo-recognition subcomplex composed of Vps26, Vps29, and Vps35. We collaborated with James Hurley and Alasdair Steven to solve the atomic structure of the Vps26-Vps29-Vps35 subcomplex. The structure revealed that Vps35 is a rod-shaped, alpha-helical solenoid onto which Vps26 and Vps29 assemble at either end of the rod. Vps26 resembles a clathrin adaptor known as arrestin, whereas Vps29 belongs to a family of metallophosphoesterases. The structure has several sites for binding to SNX proteins and cargo and can flex to conform to the curved shape of transport vesicles and tubules.

We recently found that the recruitment of the Vps26-Vps29-Vps35 subcomplex to membranes involves, in addition to the SNX subcomplex, the small GTP-binding protein Rab7. Perturbation of either the SNX subcomplex or Rab7 results in dissociation of the Vps26-Vps29-Vps35 subcomplex from membranes. Loss of retromer function leads to missorting of lysosomal enzymes and consequent accumulation of undegraded materials in lysosomes, a phenotype characteristic of lysosomal storage disorders.

Bonifacino JS, Hurley JH. Retromer. Curr Op Cell Biol 2008;20:427-436.
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;449:1063-1067.
Rojas R, Van Vlijmen T, Mardones GA, Prabhu Y, Rojas AL, Mohammed S, Heck AJR, Raposo G, van der Sluijs P, Bonifacino JS. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J Cell Biol 2008;183:513-526.

Role of the GARP complex in transport from endosomes to the Golgi complex

Pérez-Victoria, Mardones

Endosomal transport carriers formed by the action of the retromer must dock at and fuse with the trans-Golgi network in order to deliver their cargo. We recently found that GARP, a multiprotein complex originally described in yeast, plays such a role in mammalian cells. Interference with GARP blocks the delivery of cargos such as mannose 6-phosphate receptors and shiga toxin from endosomes to the trans-Golgi network, indicating that GARP plays a general role in retrograde transport. A mutation in Vps54, one of the GARP subunits, was recently identified in the Wobbler mouse mutant, an animal model of amyotrophic lateral sclerosis. We found that the Wobbler mutation does not prevent the function of GARP in retrograde transport, suggesting that the disease is likely attributable to a more subtle defect in transport or some other function of GARP.

Pérez-Victoria FJ, Mardones GA, Bonifacino JS. Requirement of the human GARP complex for mannose 6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes. Mol Biol Cell 2008;19:2350-2362.

Ubiquitin binding and conjugation regulate protein recruitment to endosomes

Mattera

Rab GTPases and ubiquitination are additional factors that regulate transmembrane cargo sorting in endocytic and lysosomal targeting pathways. The endosomal protein Rabex-5 intersects these two layers of regulation by functioning as both a guanine-nucleotide exchange factor (geF) for Rab5 and a substrate for ubiquitin (ub) binding and conjugation. this past year, we demonstrated that the ability of Rabex-5 to bind to ub is essential for its recruitment from the cytosol to endosomes, independently of its geF activity and of Rab5. We also showed that monoubiquitinated Rabex-5 is enriched in the cytosol. these observations are consistent with a model whereby a cycle of ub binding and monoubiquitination regulates the association of Rabex-5 with endosomes.

Mattera R, Bonifacino JS. Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes. EMBO J 2008;27:2484-2494.

¹Peter Yang, BS, former Postbaccalaureate Fellow
²Rosa Puertollano, PhD, former Postdoctoral Fellow

Collaborators

Michael Brenner, MD, Harvard Medical School, Boston, MA
Eric T. Everett, PhD, University of North Carolina, Chapel Hill, NC
Eric Freed, PhD, HIV Drug Resistance Program, NCI, Frederick, MD
John Guatelli, PhD, University of California San Diego, San Diego, CA
James Hurley, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Paul A. Overbeek, PhD, Baylor College of Medicine, Houston, TX
Alasdair Steven, PhD, Laboratory of Structural Biology Research, NIAMS, Bethesda, MD
Peter van der Sluijs, PhD, Utrecht University School of Medicine, Utrecht, The Netherlands

For further information, contact bonifacinoj@mail.nih.gov or visit http://cbmp.nichd.nih.gov/sipt