SFB 638:  Dynamics of Macromolecular Complexes in Biosynthetic Transport

• Subject Area: Biology
• Term: from 2004 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5485988

• Final Report Year: 2016

Final Report Abstract

A living cell exists of protein- and nuclear acid building blocks, as well as of membranes to define the cell border protecting from its exterior and defining compartments in its interior. Many of these building blocks assemble to higher ordered structures in order to form molecular machines. A functional cell then could be defined by the formation of compartments and molecular machines that is controlled with regard to time and space.On our journey to learn about functions of macromolecular assemblies in a cell by studying the dynamics of such complexes employed in steps of intracellular transport, we have made quite some progress. Our focus was on the analysis of mechanisms in transport between nucleus and cytoplasm (Part B) and on biosynthetic transport from the ribosome to the endomembrane system (part A). To this end we capitalized on the ample know how available within Heidelberg Molecular Life Sciences and specifically combined basic molecular research on viral functions with biophysical, biochemical, molecular biological and structural approaches in eukaryotic cells. Whereas at the outset our knowledge of individual building blocks as part of transport machinery was quite complete, only limited information was available as to more sophisticated assemblies of such building blocks and their dynamics, i.e. their functional interplay. In this summary two examples are given of how analyzing structures of higher assemblies combined with biochemical approaches led to novel and original insight. Most of the ca. 30 individual components of the nuclear pore were already categorized at the onset of this CRC, but their interactions and assembly into higher order complexes were poorly understood. Within the SFB, not only was the better part of the nuclear pore reconstituted from many of its individual protein components as a basis to understand its structure, but in addition unexpected functional interactions were defined that led to novel concepts of RNA transport and ribosome biogenesis. Likewise, novel concepts were reached based on studies of the mechanism of action of ribosome-associated chaperons in co-translational folding and transport of proteins. Trigger factor (TF) is the first chaperon to interact with the nascent protein chain in bacteria. TF binds and stabilizes partial protein folds while distant interactions are prevented. Ribosome proximity and TF binding limit conformational sampling of nascent chains, suggesting TF can avoid early misfolding or rescue misfolded protein species generated co-translationally. The TF interactome was assessed by ribosome profiling and revealed that most of the nascent polypeptides are clients of TF, but only after they reached about 100 aa residues. This allows enzymatic processing of nascent chains by the essential ribosome interacting enzymes peptide deformylase and methionine aminopeptidase. From studies on maturation of a hetero-dimeric model protein it was observed that assembly of its subunits occurs close to the site of synthesis, involving co-translational interactions of nascent subunits. As a novel concept a role was attributed to TF in delaying the onset of co-translational interactions until the subunit dimer interface is fully exposed on the ribosomal surface. Studying dynamics of macromolecular assemblies being in the focus of each single project has solicited a continuous mutual interest, and consequently many lively and fruitful discussions and collaborations, including intense and uninhibited exchange of materials and methods. Here, I would like to stress that the second part of our research period happened to coincide with the extreme progress of structural elucidation by cryo electron microscopy, and that inclusion of and collaboration with cryo-EM groups has proven extraordinarily useful for our scientific progress.

Publications

• (2004). Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75-86
  Susana Rodriguez-Navarro, Tamás Fischer, Ming-Juan Luo, Oreto Antunez, Susanne Brettschneider, Jose E. Perez-Ortin, Robin Reed, and Ed Hurt
  
• (2004). Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590-596
  Ferbitz, L., Maier, T., Patzelt, H., Bukau, B., Deuerling, E., and Ban, N.
  
• (2005). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med. 1(7):791-6. Epub Erratum in: Nat Med. 2005 Aug;11(8):905
  Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Kräusslich HG, Mizokami M, Bartenschlager R, Liang TJ
  
• (2005). The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8. Proc.Natl.Acad.Sci.USA 102, 17366-17371
  Hou, H., Subramanian, K., LaGrassa, T.J., Markgraf, D., Dietrich, L.E.P., Decker, N., and Ungermann, C.
  
• (2005). The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex. J. Cell Biol. 168, 401-14
  LaGrassa, T.J., and Ungermann, C.
  
• (2005). Transcriptional feedback of Neurospora circadian clock gene by phosphorylation-dependent inactivation of its transcription factor. Cell 122, 235-246
  Schafmeier, T, Haase, A, Káldi, K, Scholz, J, Fuchs, M and Brunner, M
  
• (2006). A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes. Nat Cell Biol 8, 257-263
  Bohnsack, M.T., Stüven, T., Kuhn, C., Cordes, V.C. and Görlich, D.
  
• (2006). Cell surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2. Proc. Natl. Acad Sci. U.S.A. 103:15479-15484
  Christoph Zehe, André Engling, Sabine Wegehingel, Tobias Schäfer, and Walter Nickel
  
• (2006). FG-rich repeats of nuclear pore proteins form a threedimensional meshwork with hydrogel-like properties. Science 314, 815-817
  Frey, S., Richter, R.P. and Görlich, D.
  
• (2007). A saturated FG-repeat hydrogel can reproduce the permeability-properties of nuclear pore complexes. Cell 130(3), 512-23
  Frey, S. and Görlich, D.
  
• (2007). Novel cargo-binding site in the beta- and delta-subunits of coatomer. J. Cell Biol. 179: 209-217
  Michelsen, K., Schmid, V., Metz, J., Liebel, U., Schwede, T., Spang, A., and Schwappach, B.
  
• (2008). Structural basis for specific substrate recognition by the chloroplast signal recognition particle protein cpSRP43. Science 321(5886): 253-256
  Stengel, K. F., I. Holdermann, P. Cain, C. Robinson, K. Wild and I. Sinning
  
• (2008). Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe. 4:592-9
  Carlson LA, Briggs JA, Glass B, Riches JD, Simon MN, Johnson MC, Müller B, Grünewald K, Kräusslich HG
  
• (2009). Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe. 5(4):365-75
  Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, Fuller SD, Antony C, Krijnse-Locker J, Bartenschlager R
  (See online at https://dx.doi.org/10.1016/j.chom.2009.03.007)
• (2009). HIV-1 Nef interferes with host cell motility by deregulation of cofilin. Cell Host and Microbe, 6: 174-186
  Stolp, B., Reichman-Fried, M., Abraham. L., Pan, X., Giese, S.I., Hannemann, S., Goulimari, P., Raz, E., Grosse, R. and Fackler, O.T.
  
• (2009). Spatially and kinetically resolved changes in the conformational dynamics of the Hsp90 chaperone machine. EMBO J 28, 602–613
  Graf, C, Stankiewicz, M, Kramer, G & Mayer, MP
  
• (2009). Structural insights into tail-anchored protein binding and membrane insertion by Get3. Proc Natl Acad Sci USA 106(50): 21131-21136
  Bozkurt, G., Stjepanovic, G., Vilardi, F., Amlacher, S., Wild, K., Bange, G., Favaloro, V., Rippe, K., Hurt, E., Dobberstein, B. and Sinning, I.
  
• (2009). Structure and assembly of immature HIV. Proc Natl Acad Sci USA. 106:11090-5
  Briggs JA, Riches JD, Glass B, Bartonova V, Zanetti G, Kräusslich HG
  
• (2010). Segregation of yeast nuclear pores. Nature 466, E1
  Khmelinskii, A., Keller, P. J., Lorenz, H., Schiebel, E. & Knop, M.
  (See online at https://dx.doi.org/10.1038/nature09255)
• (2010). Ubiquitylation regulates interactions of astral microtubules with the cleavage apparatus. Curr Biol. 20:1233-43
  Kammerer, D., L. Stevermann, and D. Liakopoulos
  
• (2010)Photoadaptation in Neurospora by competitive interaction of activating and inhibitory LOV domains. Cell 142, 762-772
  Malzahn, E., Ciprianidis S., Kaldi, K., Schafmeier, T. and Brunner, M.
  
• (2011). "Structural basis for the molecular evolution of SRP-GTPase activation by protein." Nat Struct Mol Biol 18(12): 1376-1380
  Bange, G., N. Kummerer, P. Grudnik, R. Lindner, G. Petzold, D. Kressler, E. Hurt, K. Wild and I. Sinning
  
• (2011). Circadian conformational change of the Neurospora clock protein FREQUENCY triggered by clustered hyperphosphorylation of a basic domain. Mol Cell 43, 713-722
  Querfurth, C., Diernfellner, A.C.R., Gin, E., Höfer, T. and Brunner M.
  
• (2011). Coatomer and dimeric ADP ribosylation factor 1 (Arf1) promote distinct steps in membrane scission. J Cell Biol. 194(5):765-77
  R. Beck, S. Prinz, P. Diestelkötter-Bachert, S. Röhling, F. Adolf, K. Hoehner, S. Welsch, P. Ronchi, B. Brügger, J.A. Briggs, and F. Wieland
  
• (2011). Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile. Cell 146, 277-289
  Amlacher Stefan, Sarges Phillip, Flemming Dirk, van Noort Vera, Kunze Ruth, Devos Damien, Arumugam Manimozhiyan, Bork Peer and Hurt Ed
  
• (2011). Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147, 1295-1308
  Oh, E., Becker, A.H., Sandikci, A., Huber, D., Chaba, R., Gloge, F., Nichols, R.J., Typas, A., Gross, C.A., Kramer, G., et al.
  
• (2011). Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex. Science 333(6043): 758-762
  Stefer, S., Reitz, S., Wang, F., Wild, K., Pang, Y. Y., Schwarz, D., Bomke, J., Hein, C., Lohr, F., Bernhard, F., Denic, V., Dotsch, V. and Sinning, I.
  
• (2012). Dynamics of the regulation of Hsp90 by the co-chaperone Sti1. EMBO J 31, 1518–1528
  Lee, C-T, Graf, C, Mayer, FJ, Richter, SM & Mayer, MP
  (See online at https://doi.org/10.1038/emboj.2012.37)
• (2012). HIV-1 Nef Interferes With T Lymphocyte Circulation Through Confined Environments in vivo. Proc. Natl. Acad. Sci. USA, 109: 18541–18546
  Stolp, B., Imle, A., Coelho, F.M., Hons, M., Mendiz, R.G., Lyck, R., Stein, J.V. and Fackler, O.T.
  (See online at https://doi.org/10.1073/pnas.1204322109)
• (2012). Molecular recognition of a single sphingolipid species by a protein’s transmembrane domain. Nature 481:525-529
  F.-X. Contreras, A.M. Ernst, P. Haberkant, P. Björkholm, E. Lindahl, B. Gönen, C. Tischer, A. Elofsson, G. von Heijne, C. Thiele, R. Pepperkok, F. Wieland, and B. Brügger
  (See online at https://doi.org/10.1038/nature10742)
• (2012). Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol Cell 48, 863-874
  Kityk, R., Kopp, J., Sinning, I., and Mayer, M. P.
  (See online at https://doi.org/10.1016/j.molcel.2012.09.023)
• (2012). Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Molecular Cell 48, 863–874
  Kityk, R, Kopp, J, Sinning, I & Mayer, MP
  (See online at https://doi.org/10.1016/j.molcel.2012.09.023)
• (2012). The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions. Science 336, 1451-1454
  Faini, M., Prinz, S., Beck, R., Schorb, M., Riches, J.D., Bacia, K., Brügger, B., Wieland, F. T. and Briggs, J.A.G.
  (See online at https://doi.org/10.1126/science.1221443)
• (2014). SRP RNA remodeling by SRP68 explains its role in protein translocation. Science 344(6179): 101-104
  Grotwinkel, J. T., K. Wild, B. Segnitz and I. Sinning
  (See online at https://doi.org/10.1126/science.1249094)
• (2015). A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly. Science 349, 195-198
  Dodonova, S.O., Diestelkoetter-Bachert, P., von Appen, A., Hagen, W.J.H., Beck R., Beck M., Wieland, F., and Briggs, J.A.G.
  (See online at https://doi.org/10.1126/science.aab1121)
• (2015). Linker Nups connect the nuclear pore complex inner ring with outer ring and transport channel. Nature Struct. Mol. Biol. 22, 774-81
  Fischer Jessica, Teimer Roman, Amlacher Stefan, Kunze Ruth and Hurt Ed
  (See online at https://doi.org/10.1038/nsmb.3084)
• (2015). Operon structure and cotranslational subunit association direct protein assembly in bacteria. Science. 350, 678-80
  Shieh, Y.W., Minguez, P., Bork, P., Auburger, J.J., Guilbride, D.L., Kramer, G., and Bukau, B.
  (See online at https://doi.org/10.1126/science.aac8171)
• (2015). Syo1 chaperones 5S-RNP assembly during ribosome biogenesis by RNA mimicry, Nat. Commun. 6: 6510
  Calviño, F.R., Kharde, S., Ori, A., Hendricks, A., Wild, K., Kressler, D., Bange, G., Hurt, E., Beck, M. & Sinning, I.
  (See online at https://doi.org/10.1038/ncomms7510)
• (2015). The fission yeast MTREC complex targets CUTs and unspliced pre-mRNAs to the nuclear exosome. Nat. Commun. 7:7050
  Zhou, Y., Zhu, J., Schermann, G., Ohle, C., Bendrin, K., Sugioka-Sugiyama, R., Sugiyama, T., and Fischer, T.
  (See online at https://doi.org/10.1038/ncomms8050)
• (2016). The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes. Nat. Comm. 7:11482
  Ritterhoff, T., Das, H., Hofhaus, G., Schröder, R.R., Flotho, A. and Melchior, F.
  (See online at https://doi.org/10.1038/ncomms11482)