Molecular machines in protein targeting and membrane protein biogenesis
Gruppenleiter: Irmgard Sinning
Prestigious medal from the State of Bavaria for Irmi Sinning
Leibniz-Prize 2014 awarded to Irmgard Sinning
Featured in nature Crystallography at 100 issue
Our goal is to understand the molecular mechanisms of key cellular processes at the atomic level. We focus on molecular machines involved in protein targeting, insertion and membrane translocation. The combination of structural biology to elucidate the three-dimensional architecture of macromolecular complexes with functional analyses provides the mechanistic principles.
The SRP pathway: Insertion of membrane proteins
Membrane proteins comprise more than 25% of the cellular proteome and their function depends on insertion into the correct target membrane. The work of Blobel and co-workers awarded with the Nobel prize in 1999 defined an intrinsic signal (the signal sequence) that is essential for governing them to the endoplasmic reticulum. They utilize predominantly the delivery pathway of the signal recognition particle (SRP). The SRP system is regulated by a unique set of GTP binding proteins which communicate with an RNA to coordinate protein synthesis at the ribosome with membrane insertion by the translocon. Our goal is to deduce the molecular and structural framework of this multistep regulatory pathway that ensures the fidelity of protein transport. We could show e.g. that SRP RNA remodeling by SRP68 in eukaryotes is required for SRP function in elongation arrest and protein translocation.
Becker, M.M.M., Lapouge, K., Segnitz, B., Wild, K. & Sinning, I. (2017) Structures of human SRP72 reveal its function in co-translational protein targeting, Nucl. Acids Res. 45: 470-481.
Wild, K., Bange, G., Motiejunas, D., Kribelbauer, J., Hendricks, A., Segnitz, B., Wade, R., & Sinning, I. (2016) Structural basis for conserved regulation and adaptation of the signal recognition particle targeting complex, J. Mol. Biol. 428: 2880-97.
Beckert, B., Kedrov, A., Sohmen, D., Kempf, G., Wild, K., Sinning, I., Stahlberg, H., Wilson, D. & Beckmann, R. (2015) Translational arrest by a prokaryotic signal recognition particle is mediated by RNA interactions, Nat. Struct. Mol. Biol. 22: 767-73.
Jadhav, B., McKenna, M., Johnson, N., High, S., Sinning, I. & Pool, M.R. (2015) Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation, Nat Comms. 6: 10133.
Horn, A., Hennig, J., Ahmed, J.L., Stier, G., Wild, K., Sattler, M. & Sinning, I. (2015) Structural basis for cpSRP43 chromodomain selectivity and dynamics in Alb3 insertase interaction, Nat Comms. 6: 8875.
Jadhav, B., Wild, K., Pool, M.R. & Sinning, I. (2015) Structure and switch cycle of SRβ as ancestral eukaryotic GTPase associated with secretory membranes, Structure 23: 1838-47.
Kempf, G., Wild, K. & Sinning, I. (2014) Structure of the complete bacterial SRP Alu domain, Nucl. Acids Res. 42: 12284-94.
Grotwinkel, J.T., Wild, K., Segnitz, B. & Sinning, I. (2014) SRP RNA remodeling by SRP68 explains its role in protein translocation, Science 344: 101-104. DOI:10.1126/science.1249094.
Bange, G. & Sinning, I. (2013) SIMIBI Twins in protein targeting and localization, Nat. Struct. Mol. Biol. 20: 776-80.
Holdermann, I., Meyer, H., Round, A., Wild, K., Sattler, M. & Sinning, I. (2012) Chromodomains read the arginine code of post-translational targeting, Nat. Struct. Mol. Biol. 19: 260-63.
The Get pathway: Insertion of tail-anchored membrane proteins
In the textbooks, insertion of membrane proteins into the ER is mediated by SRP which relies on the presence of an N-terminal signal sequence. However, a subset of membrane proteins involved in important physiological processes ranging from intracellular trafficking to protein degradation and programmed cell death lack such a signal sequence. Instead they carry their targeting signal at the C-terminus and are therefore termed tail-anchored (TA) proteins. These proteins are targeted to the ER post-translationally by the recently described GET pathway (guided entry of tail-anchored proteins). While the identification of the components seems complete, the molecular and structural framework of this pathway is far from being understood. Our goal is to fill this gap.
Denic, V., Doetsch, V. & Sinning, I. (2013) Endoplasmic reticulum Targeting and Insertion of Tail-anchored membrane proteins by the GET pathway. Cold Spring Harb Perspect Biol. 5 (8): a013334.
Stefer, S., Reitz, S., Wang, F., Wild, K., Pang, Y.Y., Schwarz, D., Bomke, J., Hein, C., Löhr, F., Bernhard, F., Denic, V., Dötsch, V. & Sinning, I. (2011) Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex. Science 333: 758-62.
Bozkurt, G., Stjepanovic, G., Vilardi, F., Amlacher, S., Wild, K., Bange, G., Favaloro, V., Rippe, K., Hurt, E., Dobberstein, B. & Sinning, I. (2009) Structural insights into tail-anchored protein binding and membrane insertion by Get3. Proc Natl Acad Sci U S A. 106: 21131-6.
Ribosome biogenesis, ribosome associated chaperones and enzymes
Ribosomes are among the largest cellular machines and carry out protein translation. However, the activity of ribosomes is not restricted to the pure synthesis of polypeptides. They are crucial players also in the modification, folding, assembly and membrane-insertion of newly-synthesized polypeptides as they provide a binding platform for various ligands and thereby determine the fate of nascent polypeptides. A number of these ligands bind near the ribosomal tunnel exit which plays a central role in the post-translational activities of the ribosome. We aim to deduce the molecular and structural framework of the processes at the ribosomal exit tunnel and focus on: Ribosome-associated chaperones and enzymes. Chaperones are essential in promoting correct protein folding in all domains of life. The ribosome-associated complex (RAC) consists of the Hsp70 member Ssz1 and its Hsp40 Zuo1, which together with Ssb form a functional entity promoting co-translational folding of nascent chains in all eukaryotes. N-terminal acetylation of nascent protein chains is one of the most common co-translational modifications in eukaryotes. Over 80% of all cytosolic proteins in mammalia and more than 40% of all proteins in the yeast Saccharomyces cerevisiae are found to be acetylated by Nα-terminal acetyltransferase (Nat) complexes. Each Nat consists of at least two subunits, i.e. a ribosomal adapter (anchor subunit) and the catalytic subunit which seem to be bound close to the ribosomal exit tunnel. Ribosome Biogenesis. Ribosomal proteins are synthesized in the cytoplasm, but eukaryotic ribosome assembly occurs predominantly in the nucleus. Ribosome biogenesis requires the ordered assembly of ribosomal proteins and ribosomal RNAs, and involves more than 150 factors. We have recently focused on 5S RNP biogenesis and characterized a novel nuclear import factor (symportin1), which ensures the coordinated and stoichiometric incorporation of Rpl5 and Rpl11 into pre-60S ribosomes.
Weyer, F. A., Gumiero, A., Valentín Gesé, G., Lapouge, K. & Sinning, I. (2017) Structural insights into a unique Hsp70/Hsp40 interaction in the eukaryotic ribosome-associated complex, Nat. Struct. Mol. Biol. 24: 144-151.
Baßler, J., Ahmed, Y.L., Kallas, M., Kornprobst, M., Rodriguez-Calvino, F., Gnädig, M., Thoms, M., Stier, G., Ismail, S., Kharde, S., Castillo, N., Griesel, S., Bastuck, S., Bradatsch, B., Thomson, E., Flemming, D., Sinning, I.* & Hurt, E.* (2017) Interaction network of the ribosome assembly machinery from a eukaryotic thermophile, Protein Science 26: 327-342.
Ohle, C., Tesorero, R., Schermann, G., Dobrev, N., Sinning, I. & Fischer, T. (2016) RNA-DNA hybrids and RNase H activity are required for efficient DSB repair, Cell 167: 1001-13.
Gumiero, A., Conz, C., Valentín Gesé, G., Zhang, Y., Weyer, F. A., Lapouge, K., Kappes, J., von Plehwe, U., Schermann, G., Fitzke, E., Wölfle, T., Fischer, T., Rospert, S. & Sinning, I. (2016) Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain, Nat. Comms. 7: 13563.
Thoms, M., Ahmed. Y.L., Maddi, K., Hurt, E. & Sinning, I. (2016) Concerted removal of the Erb1-Ytm1 complex in ribosome biogenesis relies on an elaborate interface, Nucl. Acids Res. 44: 926-39.
Kharde, S., Calviño, F.R., Gumiero, A., Wild, K. & Sinning, I. (2015) The structure of Rpf2-Rrs1 explains its role in ribosome biogenesis, Nucl. Acids Res. 43: 7083-95.
Pausch, P., Singh, U., Ahmed, Y.L., Pillet, B., Altegoer, F., Stier, G., Thoms, M., Hurt, E., Sinning, I., Bange, G. & Kressler, D. (2015) Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones, Nat. Commun. 6: 7494.
Calviño, F.R., Kharde, S., Ori, A., Hendricks, A., Wild, K., Kressler, D., Bange, G., Hurt, E., Beck, M. & Sinning, I. (2015) Syo1 chaperones 5S-RNP assembly during ribosome biogenesis by RNA mimicry, Nat. Comms. 6: 3491.
Bassler, J., Paternoga, H., Holdermann, I., Thoms, M., Grannemann, S., Barrio-Garcia, C., Nyarko, A., Stier, G., Clark, S., Schraivogel, D., Kallas, M., Beckmann, R., Tollervey, D., Barbar, E., Sinning*, I. & Hurt*, E. (2014) A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation, J. Cell Biol. 207: 481-498.
Leidig, C., Thoms, M., Holdermann, I., Bradatsch, B., Berninghausen, O., Bange, G., Sinning, I., Hurt, E. & Beckmann, R. (2014) 60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle. Nat. Commun. 5: 3491.
Leidig, C., Bange, G., Kopp, J., Amlacher, S., Aravind, A., Wickles, S., Witte, G., Hurt, E., Beckmann, R. & Sinning, I. (2013) Structural characterization of a eukaryotic co-translational chaperone, the ribosome-associated complex (RAC). Nat. Struct. Mol. Biol. 20: 23-8.
Kressler, D., Bange, G. Ogawa, Y., Bradatsch, B., Pratte, D., Strauß, D., Amlacher, S., Yoneda,Y., Katahira, J., Sinning*, I., & Hurt*, E. (2012) Synchronizing nuclear import of ribosomal proteins with ribosome assembly. Science 338: 666-71.
Membrane insertases and membrane protein production
Membrane proteins preferentially use the SecYEG translocation channel for membrane insertion. However, membrane proteins that play important roles in respiration and energy transduction mechanisms in bacteria, mitochondria and chloroplasts are inserted and assembled by the YidC/Oxa1/Alb3 family of membrane insertases. We have recently focussed on the link between Alb3 and chloroplast SRP system (cpSRP). For the production of membrane proteins such as G protein coupled receptors and transporters, we have developed a novel expression system using transgenic Drosophila. We exploit the naturally abundant membrane stacks of the photoreceptor cells and noticed the superior quality of the expressed proteins compared to conventional expression systems. One of our interests is the regulation of membrane proteins by lipids.
Hackmann, Y., Jödicke, L., Panneels, V. & Sinning, I. (2015) Expression of membrane proteins in the eyes of transgenic Drosophila melanogaster. Methods Enzymol. 556: 219-39.
Panneels, V., Kock, I., Krijnse-Locker, J., Rezgaoui, M. & Sinning, I. (2011) Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels. PLoS One 6: e18478.
Panneels, V. & Sinning, I. (2010) Overexpression of membrane proteins in fly eyes. In: Heterologous Expression of Membrane Proteins: Methods and Protocols, Series: Methods in Molecular Biology, Humana Press (I. Mus-Veteau, ed.). Methods Mol. Biol. 601:135-47.
OUR APPROACHWe study model systems from all three domains of life to understand our target machines from an evolutionary perspective. The combination of cell biology and biophysics allows to unravel the molecular mechanisms of these machines and their regulation in the context of a living cell. We use advanced X-ray crystallography techniques as our key method – together with biochemical methods which are established in the lab or in long-standing collaborations (e.g. ITC, UV and CD spectroscopy, amide hydrogen deuterium exchange with mass spectrometry, EPR spectroscopy, cryo-electron microscopy, SAXS, NMR). State of the art expression and purification techniques are the basis of our studies. We have established a HTP-crystallization platform that is open to external users.
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