62 research outputs found
Structural basis for the binding of tryptophan-based motifs by ÎŽ-COP.
Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding ÎČγΎζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αÎČ'Δ-COP B-subcomplex. We present the structure of the C-terminal ÎŒ-homology domain of the yeast ÎŽ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP ÎŒ subunits to bind YxxΊ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to ÎŽ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian ÎŽ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that ÎŽ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.We should like to thank the beamline scientists at the Diamond Light Source and Mike Lewis (MRC LMB), Gerry Johnston (Dalhousie University), and Mark Rose (Princeton University) for helpful discussions and technical advice. RJS and DJO are funded by a Wellcome Trust fellowship to DJO (090909). PPP was funded by Canadian Institute of Health Research. RD acknowledges support from the DFG Excellence Cluster âInflammation and Interfacesâ (ECX306) and the University of LĂŒbeck. SMT and FMH acknowledge support from NIH (GM071574). PRE is funded by MRC grant U105178845This is the author accepted manuscript. The final version is available from PNAS via http://dx.doi.org/10.1073/pnas.150618611
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The structural basis of rubisco phase separation in the pyrenoid
Approximately one-third of global CO2 fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle
Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion
SM proteins stabilize cis-SNARE complexes leading to a specific preferred topology for trans-SNARE formation
Molecular architecture of the complete COG tethering complex
The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion
Membrane fusion: Structure snared at last
AbstractThe structure of the core of the neuronal âSNARE complexâ, involved in neurotransmitter release, has been determined recently. Its topological similarity to viral fusion proteins suggests how the SNARE complex might facilitate membrane fusion
Ernest Renan : la science, la métaphysique, la religion et la question de leur avenir
On croit souvent que, pour Renan, lâavenir appartiendrait Ă la seule science ; la religion nâen aurait, au contraire, Ă peu prĂšs aucun. Mais mĂȘme un lecteur simplement superficiel ne tarde cependant pas Ă se rendre compte que sa position est bien diffĂ©rente. La prĂ©face du PrĂȘtre de Nemi (un drame philosophique quâil a publiĂ© en 1885) commence de la façon suivante : « Jâai voulu, dans cet ouvrage, dĂ©velopper une pensĂ©e analogue Ă celle du messianisme hĂ©breu, câest-Ă -dire la foi au triomphe dĂ©f..
The COG and COPI Complexes Interact to Control the Abundance of GEARs, a Subset of Golgi Integral Membrane Proteins
The conserved oligomeric Golgi (COG) complex is a soluble hetero-octamer associated with the cytoplasmic surface of the Golgi. Mammalian somatic cell mutants lacking the Cog1 (ldlB) or Cog2 (ldlC) subunits exhibit pleiotropic defects in Golgi-associated glycoprotein and glycolipid processing that suggest COG is involved in the localization, transport, and/or function of multiple Golgi processing proteins. We have identified a set of COG-sensitive, integral membrane Golgi proteins called GEARs (mannosidase II, GOS-28, GS15, GPP130, CASP, giantin, and golgin-84) whose abundances were reduced in the mutant cells and, in some cases, increased in COG-overexpressing cells. In the mutants, some GEARs were abnormally localized in the endoplasmic reticulum and were degraded by proteasomes. The distributions of the GEARs were altered by small interfering RNA depletion of Δ-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both Δ-COP and either Cog1 or Cog2. COG and COPI may work in concert to ensure the proper retention or retrieval of a subset of proteins in the Golgi, and COG helps prevent the endoplasmic reticulum accumulation and degradation of some GEARs
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