NORMAL GENOMIC

• Kurz T et al.
• Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation.
• Mol Cell. 2008; 29: 23-35
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• Cullin-based E3 ubiquitin ligases are activated through modification ofthe cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulatescullin neddylation and thus ubiquitin ligase activity. Here we describethe 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminalubiquitin-binding (UBA) domain and a C-terminal domain of uniquearchitecture, which we termed PONY domain. A conserved surface on Dcn1 isrequired for direct binding to cullins and for neddylation. The reciprocalbinding site for Dcn1 on Cdc53 is located approximately 18 A from the siteof neddylation. Dcn1 does not require cysteine residues for catalyticfunction, and directly interacts with the Nedd8 E2 Ubc12 on a surface thatoverlaps with the E1-binding site. We show that Dcn1 is necessary andsufficient for cullin neddylation in a purified recombinant system. Takentogether, these data demonstrate that Dcn1 is a scaffold-like E3 ligasefor cullin neddylation.

• Seigneuret M
• Complete predicted three-dimensional structure of the facilitatortransmembrane protein and hepatitis C virus receptor CD81: conserved andvariable structural domains in the tetraspanin superfamily.
• Biophys J. 2006; 90: 212-27
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• Tetraspanins are a superfamily of transmembrane proteins implicated incellular development, motility, and activation through their interactionswith a large range of proteins and with specific membrane microdomains.The complete three-dimensional structure of the tetraspanin CD81 has beenpredicted by molecular modeling and from the crystallographic structure ofthe EC2 large extracellular domain. Periodicity of sequence conservation,homology modeling, secondary structure prediction, and protein dockingwere used. The transmembrane domain appears organized as a four-strandedleft-handed coiled coil directly connecting to two helices of the EC2. Asmaller extracellular loop EC1 contains a small largely hydrophobicbeta-strand that packs in a conserved hydrophobic groove of the EC2. Thepalmitoylable intracellular N-terminal segment forms an amphipathicmembrane-parallel helix. Structural variability occurs mainly in anhypervariable subdomain of the EC2 and in intracellular regions.Therefore, the variable interaction selectivity of tetraspanins originatesboth from sequence variability within structurally conserved domains andfrom the occurrence of small structurally variable domains. In CD81 andother tetraspanins, the numerous membrane-exposed aromatic residues areasymmetrically clustered and protrude on one side of the transmembranedomain. This may represent a functional specialization of these two sidesfor interactions with cholesterol, proteins, or membrane microdomains.

• Dennes A et al.
• The novel Drosophila lysosomal enzyme receptor protein mediates lysosomalsorting in mammalian cells and binds mammalian and Drosophila GGAadaptors.
• J Biol Chem. 2005; 280: 12849-57
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• Biogenesis of lysosomes depends in mammalian cells on the specificrecognition and targeting of mannose 6-phosphate-containing lysosomalenzymes by two mannose 6-phosphate receptors (MPR46, MPR300), keycomponents of the extensively studied receptor-mediated lysosomal sortingsystem in complex metazoans. In contrast, the biogenesis of lysosomes ispoorly investigated in the less complex metazoan Drosophila melanogaster.We identified the novel type I transmembrane protein lysosomal enzymereceptor protein (LERP) with partial homology to the mammalian MPR300encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats thatshare homology to the 15 lumenal repeats found in all identified MPR300.Four of the repeats display the P-lectin type pattern of conservedcysteine residues. However, the arginine residues identified to beessential for mannose 6-phosphate binding are not conserved. Therecombinant LERP protein was expressed in mammalian cells and displayed anintracellular localization pattern similar to the mammalian MPR300. TheLERP cytoplasmic domain shows highly conserved interactions withDrosophila and mammalian GGA adaptors known to mediate Golgi-endosometraffic of MPRs and other transmembrane cargo. Moreover, LERP rescuesmissorting of soluble lysosomal enzymes in MPR-deficient cells, givingstrong evidence for a function that is equivalent to the mammaliancounterpart. However, unlike the mammalian MPRs, LERP did not bind to themultimeric mannose 6-phosphate ligand phosphomannan. Thus ligandrecognition by LERP does not depend on mannose 6-phosphate but may dependon a common feature present in mammalian lysosomal enzymes. Our dataestablish a potential important role for LERP in biogenesis of Drosophilalysosomes and suggest a GGA function also in the receptor-mediatedlysosomal transport system in the fruit fly.

• Amor JC et al.
• The structure of RalF, an ADP-ribosylation factor guanine nucleotideexchange factor from Legionella pneumophila, reveals the presence of a capover the active site.
• J Biol Chem. 2005; 280: 1392-400
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• The Legionella pneumophila protein RalF is secreted into host cytosol viathe Dot/Icm type IV transporter where it acts to recruit ADP-ribosylationfactor (Arf) to pathogen-containing phagosomes in the establishment of areplicative organelle. The presence in RalF of the Sec7 domain, present inall Arf guanine nucleotide exchange factors, has suggested thatrecruitment of Arf is an early step in pathogenesis. We have determinedthe crystal structure of RalF and of the isolated Sec7 domain and foundthat RalF is made up of two domains. The Sec7 domain is homologous tomammalian Sec7 domains. The C-terminal domain forms a cap over the activesite in the Sec7 domain and contains a conserved folding motif, previouslyobserved in adaptor subunits of vesicle coat complexes. The importance ofthe capping domain and of the glutamate in the "glutamic finger,"conserved in all Sec7 domains, to RalF functions was examined using threedifferent assays. These data highlight the functional importance ofdomains other than Sec7 in Arf guanine nucleotide exchange factors tobiological activities and suggest novel mechanisms of regulation of thoseactivities.

• Zhai P et al.
• The interaction of the human GGA1 GAT domain with rabaptin-5 is mediatedby residues on its three-helix bundle.
• Biochemistry. 2003; 42: 13901-8
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• GGA proteins regulate clathrin-coated vesicle trafficking by interactingwith multiple proteins during vesicle assembly. As part of this process,the GAT domain of GGA is known to interact with both ARF and Rabaptin-5.Particularly, the GAT domains of GGA1 and -2, but not of GGA3,specifically bind with a coiled-coil region of Rabaptin-5. Rabaptin-5interacts with Rab5 and is an essential component of the fusion machineryfor targeting endocytic vesicles to early endosomes. The recentlydetermined crystal structure of the GGA1 GAT domain has provided insightsinto its interactions with partner proteins. Here, we describe mutagenesisstudies on the GAT-Rabaptin-5 interaction. The results demonstrate that ahydrophobic surface patch on the C-terminal three-helix bundle motif ofthe GAT domain is directly involved in Rabaptin-5 binding. A GGA3-likemutation, N284S, in this Rabaptin-5 binding patch of GGA1 led to a reducedlevel of Rabaptin-5 binding. Furthermore, a reversed mutation, S293N, inGGA3 partially establishes Rabaptin-5 binding ability in its GAT domain.These results provide a structural explanation for the binding affinitydifference among GGA proteins. The current results also suggest that thebinding of GAT to Rabaptin-5 is independent of its interaction with ARF.

• Masson N, Willam C, Maxwell PH, Pugh CW, Ratcliffe PJ
• Independent function of two destruction domains in hypoxia-induciblefactor-alpha chains activated by prolyl hydroxylation.
• EMBO J. 2001; 20: 5197-206
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• Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha(HIF-alpha) subunits plays a central role in regulating transcriptionalresponses to hypoxia. Recent studies have defined a key function for thevon Hippel-Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in thisprocess, and have defined an interaction with HIF-1 alpha that isregulated by prolyl hydroxylation. Here we show that two independentregions within the HIF-alpha oxygen-dependent degradation domain (ODDD)are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolylhydroxylation. In a series of in vitro and in vivo assays, we demonstratethe independent and non-redundant operation of each site in regulation ofthe HIF system. Both sites contain a common core motif, but differ both inoverall sequence and in the conditions under which they bind to the VHLE3ligase complex. The definition of two independent destruction domainsimplicates a more complex system of pVHL-HIF-alpha interactions, butreinforces the role of prolyl hydroxylation as an oxygen-dependentdestruction signal.