Protein Biogenesis and Function


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Tilman Maier og Lars Jessen
Protein folding and Degradation
The abundance of general chaperones and proteases suggests that cells distinguish between proteins which can be refolded and "hopeless" cases fated to enter the proteolytic pathway. We are interested in the mechanisms controlling this key metabolic decision by studying various protein folding catalysts, proteases and their native substrates.

Recent references
Ehrmann, M and T. Clausen. Proteolysis as a regulatory mechanism. 2004 Annu. Rev. Genet. 38: 709-724 entrez/medline

Wilken, C., Kitzing, K., Kurzbauer, R., Ehrmann, M. and T. Clausen 2004. Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease domain. Cell 117:483-494 entrez/medline
Highlighted in Cell 2004 117:417-419

Clausen, T, Southan, C. and Ehrmann, M 2002. The HtrA family of proteases, implications for protein composition and cell fate. Mol. Cell, 10:443-455 entrez/medline

Krojer, T, Garrido-Franco, M, Huber, R., Ehrmann, M and Clausen, T. 2002. Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature, 416: 455-459 entrez/medline

Uhland, K., Mondigler, M., Spiess, C., Prinz, W., and M. Ehrmann 2000. The determinants of translocation and folding of TreF, a trehalase of E. coli. J. Biol. Chem. 275: 23439-23445 entrez/medline

Spiess, C., Beil, A., and M. Ehrmann 1999. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97: 339-347 entrez/medline
Highlighted in Nature Science Update

Membrane protein biogenesis and function
A long standing interest is the biogenesis, that is topology, membrane insertion, folding, and function of complex integral cytoplasmic mem- brane proteins. The ABC maltose transporter is used as a model to understand targeting to the cytoplasmic membrane, folding after mem- brane insertion and finally the mechanism of active transport. To address these fundamental questions we are using a mix of genetic, biochemical and biophysical techniques.

Recent references/Secretion & Membrane protein biogenesis
Eser, M., and M. Ehrmann. 2003. SecA-dependent quality control of intracellular protein localisation. Proc. Natl. Acad. Sci. USA 100: 13231-13234 entrez/medline
Highlighted in Science 302:1296
& in J. Cell Biol. 163:691

Guan, L., Ehrmann, M., Yoneyama, H., and T. Nakae 1999. Membrane Topology of the Xenobiotic-Exporting Subunit, MexB, of the MexA, B-OprM Extrusion Pump in Pseudomonas aeruginosa. J. Biol. Chem. 274: 10517-10522 entrez/medline

Melchers, K., Buhmann, A., Schuhmacher, A., Weizenegger, K., Belin, D., Grau, S., and M. Ehrmann 1999. Membrane topology of the CadA-homologous P type ATPase of Helicobacter pylori by expression of phoA fusions in Escherichia coli and the positive inside rule. Res. Microbiol. 150: 507-520 entrez/medline

Prinz, W. A., Boyd, D., Ehrmann, M., and J. Beckwith 1998. The protein translocation apparatus contributes to determining the topology of an integral membrane protein in E. coli. J. Biol. Chem. 273: 8419-8424 entrez/medline

Uhland K., Zander T., and M. Ehrmann 1995. Synthetic competition between cytoplasmic folding and translocation of a soluble membrane protein domain. Res. Microbiol. 146: 121-128. entrez/medline

Whitley P., Zander T., Ehrmann M., Haardt M., Bremer E., and G. von Heijne 1994. Sec-independent translocation of a 100-residue periplasmic N-terminal tail in the E. coli inner membrane protein ProW. EMBO J. 13: 4653-4661. entrez/medline

Uhland K., Ehrle R., Zander T. and Ehrmann, M. 1994. Requirements for translocation of periplasmic domains in polytopic membrane proteins. J. Bacteriol. 176: 4565-4571. entrez/medline

McGovern, K., Ehrmann, M., and J. Beckwith. 1991. Decoding Signals for Membrane Protein Assembly Using Alkaline Phosphatase Fusions. EMBO J. 10: 2773-2782. entrez/medline

Ehrmann, M., and J. Beckwith. 1991. Proper Insertion of Complex Membrane Protein in the Absence of its Amino-terminal Export Signal. J. Biol. Chem. 266: 16530-16533. entrez/medline

Ehrmann, M., D. Boyd & J. Beckwith. 1990. Genetic Analysis of Membrane Protein Topology Using a Novel Gene Fusion Approach. Proc. Natl. Acad. Sci. USA 87: 7574-7578. entrez/medline

Recent references/Mechanism of ABC transport
Ehrle, R., Mikhaleva, N., Boyd, D., Davidson, A.L. and Ehrmann, M. 2003 Context-dependent effects of charged residues in transmembrane segments of MalF-PhoA fusions. Res. Microbiol. 154:654-657entrez/medline

Steinke, A., Grau, S., Davidson A., Hofmann, E., and M Ehrmann 2001. Characterization of transmembrane domains 3, 4, and 5 of MalF by mutational analysis. J. Bacteriol. 183:375-381entrez/medline

Ehrmann, M., Ehrle, R., Hofmann, E., Boos, W., and A. Schloesser 1998. The ABC maltose transporter. Mol. Microbiol. 29: 685-694 entrez/medline

Ehrle R., Pick C., Ulrich R., E. Hofmann and M. Ehrmann. 1996. Characterization of transmembrane domains 6, 7 and 8 of MalF by mutational analysis. J. Bacteriol. 178: 2255-2262. entrez/medline

Target Directed Proteolysis
To allow the use of proteases as a tool in vivo we developed a new method, Target Directed Proteolysis (TDP). TDP uses Tobacco Etch Virus NIa (TEV) protease, which recognises a seven amino acid consensus sequence. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. This new method is applied to inactivate essential proteins, to study the structure and function of membrane proteins and to address questions related to protein export.

Recent references
Faber, K.N., Kram, AM., Ehrmann, M., and Veenhuis, M. 2001. The use of the Tobacco Etch Virus (TEV-) protease as a tool to determine the topology of peroxisomal membrane proteins in vivo. J. Biol. Chem. 276: 36501-36507 entrez/medline

Herskovits, A.A., Seluanov, A., Rajsbaum, R., ten Hagen-Jongman, C.M., Henrichs, T., Bochkareva, E.S., Phillips, G.J., Probst, F.J., Nakae, T., Ehrmann , M., Luirink, J., and Bibi, E. 2001. Evidence for coupling of membrane-targeting and function of the signal recognition particle-receptor FtsY. EMBO Reports 2: 1040-1046 entrez/medline

Ehrmann, M., Bolek, P., Mondigler, M., Boyd, D., and R. Lange 1997. TnTIN and TnTAP: mini-transposons for site specific proteolysis in vivo. Proc. Natl. Acad. Sci. USA 94:13111-13115 entrez/medline

Mondigler M. and M. Ehrmann. 1996. Site specific proteolsis of the E. coli SecA Protein in vivo. J. Bacteriol. 178: 2986-2988. entrez/medline

Expression of human genes in Escherichia coli
Simple in vivo systems are developed to study proteins involved in human diseases that are based on protein folding problems. Recombinant expression of the relevant human genes in E. coli is used to set up reporter systems for the early stages of e.g. amyloidosis.

Recent references
Harnasch, M., Grau, S., Dove, S., Hochschild, N., Iskandar, M-K., Xia, W. and M. Ehrmann. 2004. Bacterial expression and two-hybrid systems for human membrane proteins: Characterisation of presenilin/amyloid precursor interaction Mol. Membrane Biol. 21:373-383