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ABSTRACT: Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region.
Nature Communications 05/2013; 4:1849. · 7.40 Impact Factor
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ABSTRACT: Atg13 is a subunit of the Atg1 complex that is involved in autophagy. The middle and C-terminal regions of Atg13 are intrinsically disordered and rich in regulatory phosphorylation sites. Thus far, there have been no structural data for any part of Atg13, and no function assigned to its N-terminal domain. We crystallized this domain, and found that it has a HORMA (Hop1, Rev7, Mad2) fold. We showed that the Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PtdIns) 3-kinase subunit Atg14, but is not required for Atg1 interaction or Atg13 recruitment to the PAS. The HORMA domain of Atg13 is similar to the closed conformation of the spindle checkpoint protein Mad2. A pair of conserved arginines was identified in the structure, and tested functionally in yeast. These residues are important for autophagy, as mutations abrogate autophagy and block Atg14 recruitment. The location of these Arg residues in the structure suggests that the Atg13 HORMA domain could act as a phosphorylation-dependent conformational switch.
Autophagy 05/2013; 9(7). · 7.45 Impact Factor
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ABSTRACT: Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the "safety belt" conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.
Proceedings of the National Academy of Sciences 03/2013; · 9.68 Impact Factor
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ABSTRACT: The clathrin-associated adaptor protein (AP) complexes AP-1 and AP-2 are two members of a family of heterotetrameric assemblies that connect transmembrane protein cargo to vesicular coats. Cargo binding by AP-1 is activated by the small GTPase Arf1, while AP-2 is activated by the phosphoinositide PI(4,5)P(2) . The structures of both AP-1 and AP-2 have been determined in their locked and unlocked conformations. The structures show how different activators use different mechanisms to trigger similar large scale conformational rearrangements. The details of these mechanisms show how membrane docking and allosteric activation of AP complexes are intimately connected.
Protein Science 02/2013; · 2.80 Impact Factor
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ABSTRACT: AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the β1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.
Cell 02/2013; 152(4):755-67. · 32.40 Impact Factor
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ABSTRACT: Tyrosine-based signals fitting the YXXΦ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3 and μ4 subunits of the corresponding AP-1, AP-2, AP-3 and AP-4 complexes. Previous X-ray crystallographic analyses identified distinct binding sites for YXXΦ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXΦ signals by other members of the μ family, we solved the crystal structure at 1.85 Angstrom resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXΦ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXΦ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXΦbinding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXΦ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXΦ signals with 14-19 μM affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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ABSTRACT: Macroautophagy is a bulk clearance mechanism in which the double-membraned phagophore grows and engulfs cytosolic material. In yeast, the phagophore nucleates from a cluster of 20-30 nm diameter Atg9-containing vesicles located at a multiprotein assembly known as the preautophagosomal structure (PAS). The crystal structure of a 2:2:2 complex of the earliest acting PAS proteins, Atg17, Atg29, and Atg31, was solved at 3.05 Å resolution. Atg17 is crescent shaped with a 10 nm radius of curvature. Dimerization of the Atg17-Atg31-Atg29 complex is critical for both PAS formation and autophagy, and each dimer contains two separate and complete crescents. Upon induction of autophagy, Atg17-Atg31-Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore. The C-terminal EAT domain of Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles. These data suggest a structural mechanism for the organization of Atg9 vesicles into the early phagophore.
Cell 12/2012; · 32.40 Impact Factor
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ABSTRACT: Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PI(4,5)P(2). These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYP(X)(n)L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system.
Proceedings of the National Academy of Sciences 10/2012; 109(42):16928-33. · 9.68 Impact Factor
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ABSTRACT: The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.
PLoS Computational Biology 10/2012; 8(10):e1002736. · 5.22 Impact Factor
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ABSTRACT: The key autophagic lipid sensors are Atg18 in yeast and the WIPI proteins in mammals. Atg18 and the WIPIs belong to the PROPPIN family of proteins. PROPPINs are seven- bladed β-propellers that bind to phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. In order to understand how PROPPINs bind phosphoinositides, we have determined the crystal structure of a representative, biochemically tractable PROPPIN, Hsv2 of Kluveromyces lactis. The structure revealed that PROPPINs contain two phosphoinositide binding sites which cooperate with a hydrophobic anchoring loop in membrane binding. These three binding elements cooperate in function, as demonstrated by the incremental loss of function in Atg18 mutants impaired in combinations of the two phosphoinositide binding sites and the hydrophobic loop.
Autophagy 09/2012; 8(12). · 7.45 Impact Factor
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ABSTRACT: The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals. The clusters only form when cholesterol is included in the lipid mixture at >10 mol %. The clusters induce the formation of ordered membrane domains that exclude the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate. These results show that ESCRT complexes are capable of inducing lateral lipid phase separation under conditions where the lipids themselves do not spontaneously phase-separate. This property could facilitate ESCRT-mediated membrane budding.
Journal of Biological Chemistry 06/2012; 287(33):28144-51. · 4.77 Impact Factor
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ABSTRACT: Macroautophagy is essential to cell survival during starvation and proceeds by the growth of a double-membraned phagophore, which engulfs cytosol and other substrates. The synthesis and recognition of the lipid phosphatidylinositol 3-phosphate, PI(3)P, is essential for autophagy. The key autophagic PI(3)P sensors, which are conserved from yeast to humans, belong to the PROPPIN family. Here we report the crystal structure of the yeast PROPPIN Hsv2. The structure consists of a seven-bladed β-propeller and, unexpectedly, contains two pseudo-equivalent PI(3)P binding sites on blades 5 and 6. These two sites both contribute to membrane binding in vitro and are collectively required for full autophagic function in yeast. These sites function in concert with membrane binding by a hydrophobic loop in blade 6, explaining the specificity of the PROPPINs for membrane-bound PI(3)P. These observations thus provide a structural and mechanistic framework for one of the conserved central molecular recognition events in autophagy.
Molecular cell 06/2012; 47(3):339-48. · 14.61 Impact Factor
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ABSTRACT: The ESCRT-I and ESCRT-II supercomplex induces membrane buds that invaginate into the lumen of endosomes, a process central to the lysosomal degradation of ubiquitinated membrane proteins. The solution conformation of the membrane-budding ESCRT-I-II supercomplex from yeast was refined against small-angle X-ray scattering (SAXS), single-molecule Förster resonance energy transfer (smFRET), and double electron-electron resonance (DEER) spectra. These refinements yielded an ensemble of 18 ESCRT-I-II supercomplex structures that range from compact to highly extended. The crescent shapes of the ESCRT-I-II supercomplex structures provide the basis for a detailed mechanistic model, in which ESCRT-I-II stabilizes membrane buds and coordinates cargo sorting by lining the pore of the nascent bud necks. The hybrid refinement used here is general and should be applicable to other dynamic multiprotein assmeblies.
Structure 05/2012; 20(5):874-86. · 6.35 Impact Factor
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ABSTRACT: BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1
is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted,
BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter.
The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes
were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended
structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within
the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course
of tubular endosome biogenesis and sorting.
Journal of Biological Chemistry 02/2012; 287(8):5882-5890. · 4.77 Impact Factor
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ABSTRACT: MVB12-associated β-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-Å atomic resolution crystal structure of the MVB12B MABP domain reveals a β-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.
Proceedings of the National Academy of Sciences 02/2012; 109(6):1901-6. · 9.68 Impact Factor
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ABSTRACT: Exosomes have a growing inventory of functions, but the mechanism of protein sorting into exosomes has been unclear. Now, a signal sequence first described in viral budding provides just such a cargo sorting mechanism, revealing closer-than-expected parallelism between exosome biogenesis and the ESCRT-dependent endolysosomal pathway.
Nature Cell Biology 01/2012; 14(7):654-5. · 19.49 Impact Factor
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Nature Structural & Molecular Biology 01/2012; 19(7):661-2. · 12.71 Impact Factor
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ABSTRACT: BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.
Journal of Biological Chemistry 12/2011; 287(8):5882-90. · 4.77 Impact Factor
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ABSTRACT: Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.
Current Opinion in Structural Biology 12/2011; 21(6):785-91. · 9.42 Impact Factor
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James H Hurley
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ABSTRACT: Recruitment of the K63-linkage specific deubiquitinating enzyme AMSH is an important step in ESCRT-dependent membrane protein sorting. In this issue of Structure, Solomons et al. now reveal an extraordinarily high affinity complex between the "MIM4" region of one ESCRT-III subunit, CHMP3, and the MIT domain of AMSH.
Structure 08/2011; 19(8):1033-5. · 6.35 Impact Factor
