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Interactions that drive Sec-dependent bacterial protein transport.

ABSTRACT: Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported via other pathways. While targeting signals for each route have common overall characteristics, individual signal peptides vary greatly in their amino acid sequences. How do these diverse signals interact specifically with the proteins that comprise the appropriate transport machinery and, at the same time, avoid targeting to an alternate route? The recent publication of the crystal structures of components of the Sec transport machinery now allows a more thorough consideration of the interactions of signal sequences with these components.

SUBMITTER: Rusch SL

PROVIDER: S-EPMC2675607 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Interactions that drive Sec-dependent bacterial protein transport.

Rusch Sharyn L SL Kendall Debra A DA

Biochemistry 20070803 34

Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported via ...[more]

PMID: 17676771

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Project description:BACKGROUND:In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS:High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS:We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

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Interactions that drive Sec-dependent bacterial protein transport.

Publications

Interactions that drive Sec-dependent bacterial protein transport.

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