Protein Secretion
 

Core features of the major mechanism for the translocation of proteins across membranes are conserved throughout biology. The bacterium Escherichia coli, like all other organisms, synthesizes secreted proteins with amino-terminal signal sequences, which direct such proteins to a translocation machinery located in the cytoplasmic membrane. In this laboratory, we have used genetic approaches to identify several of the protein components of this machinery. We are currently studying the functions in this process of membrane proteins that play a key role in protein export. In particular, we are seeking mutants that alter secretion so that the transport machinery remains in the pore-open conformation. Further, we are studying the role of two proteins, SecD and SecF, which may play a role late in the translocation process. Finally, we are examining the role of features of the amino acid sequence of secreted proteins other than the signal sequence that allow such proteins to pass through the membrane.

 Sec Model

Sebastian Ahrens and Markus Eser

One project in the Beckwith laboratory tries to further our understanding of protein export across the cytoplasmic membrane of E. coli. The major E. coli protein export machinery is the Sec machinery, with its core proteins SecYEG residing within the cytoplasmic membrane. Proteins that are designated to the cell envelope are synthesized in the cytoplasm as pre-proteins with N-terminal signal sequences. Signal sequences are short (about 18 amino acids) and show little sequence homology). Preproteins are recognized by parts of the export machinery and translocation is initiated either after (post-translational) or during protein synthesis (co-translational). Initiation of co-translational translocation requires the so-called signal recognition particle (SRP). Our laboratory has identified a distinct set of signal sequences that depend on the SRP, enabling us to find a trend in the characteristics of these SRP-dependent signal sequences that we think is critical for the discrimination by the SRP: SRP-dependent signal sequences generally show higher hydrophobicity than non-SRP-dependent signal sequences. I'm working on refining our model for SRP-dependent signal sequences allowing us to distinguish between SRP-dependent and non-SRP-dependent signal sequences. As a model system we are using the export of thioredoxin 1 (TrxA) to the periplasm by the Sec machinery. TrxA, natively a cytoplasmic protein can only be exported efficiently when fused to a SRP-dependent signal sequence. In our studies we found signal sequences that are very hydrophobic but do not promote the export of TrxA. I want to use these highly hydrophobic non-SRP-signal sequences to try to find mutations by random mutagenesis that allow efficient export of TrxA. We can easily isolate mutants that lead to TrxA export by a genetic selection. Once mutant signal sequences are found, we hope to broaden our knowledge on how a subset of short sequences with little sequence homology is able to efficiently designate the mode of translocation across the cytoplasmic membrane.

People currently involved in this project:

Sebastian Ahrens

Recent Publications:

Or, E., Boyd, D., Gon, S., Beckwith, J., and Rapoport, T. The bacterial ATPase SecA functions as a monomer in protein translocation.  J. Biol. Chem. 280:9097-9105 (2005).

Huber, D., Boyd, D., Xia, Y., Olma, M.H., Gerstein, M., and Beckwith, J. Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation.  J. Bacteriol. 187:2983-2991 (2005).

Belin, D., Guzman, L.-G., Bost, S., Konakova, M., Silva, F., and Beckwith, J.  Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors.  J. Mol. Biol. 335:437-453 (2004).

Schierle CF, Berkmen M, Huber D, Kumamoto C, Boyd D, Beckwith J. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. (2003). Abstract. Paper.

Tian H, Beckwith J. Genetic Screen Yields Mutations in Genes Encoding All Known Components of the Escherichia coli Signal Recognition Particle Pathway. J Bacteriol. 184:111-118. (2002). Abstract. Paper.

Tian H, Boyd D, Beckwith J. A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery. Proc. Natl. Acad. Sci. USA. 97:4730-4735. (2000). Abstract. Paper.

Debarbieux L, Beckwith J. On the functional interchangeability, oxidant vs. reductant, of members of the thioredoxin superfamily. J. Bacteriol. 182:723-727. (2000). Abstract. 

Debarbieux L, Beckwith J. The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc. Natl. Acad. Sci. U S A. 95:10751-10756 (1998) Abstract. Paper.

Pohlschröder, M., Prinz, W., Hartmann, E., and Beckwith, J. Protein translocation in the three domains of life: variations on a theme. Cell 91:563-566 (1997) Review. No abstract available.

Prinz, W.A., Spiess, C., Ehrmann, M., Schierle, C., and Beckwith, J. Targeting of signal sequenceless proteins for export in E. coli with altered protein translocase. EMBO J. 15:5209-5217 (1996). Abstract.

Economou, A., Pogliano, J., Beckwith, J., Oliver, D.B., and Wickner, W. SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF. Cell 83:1171-1182 (1995). Abstract