Protein secretion mechanisms

Jan Tommassen

Gram-negative bacteria have developed several pathways for the secretion of proteins across the cell envelope into the extracellular milieu (1). We focus on the type II secretion system of Pseudomonas aeruginosa and the autotransporter mechanism inNeisseria meningitidis. Both systems are two-step pathways, i.e. the proteins are first transported across the inner membrane, and the periplasmic intermediate is subsequently transported across the outer membrane in a separate step. In the type II pathway, proteins have to fold into their native conformation, before they are recognized by a complex apparatus consisting of at least 12 different proteins required for transport across the outer membrane. We are interested in the mechanisms of assisted protein folding in the periplasm, the recognition of the exoproteins by the secretion machinery, and the function of the individual components and the structure of the secretion machinery. Additionally, we study the system for biotechnological applications. Particularly, we try to exploit the system for the efficient secretion of lipases and other high-value added enzymes. We study type II secretion also in Pseudomonas putida, a non-pathogen, which is advantageous for biotechnological applications. Recently, we described a new type II secretion system in this organism, which deviates strongly from the archetype type II systems (3).

In the autotransporter mechanism, the second step in the secretion process is mediated by a C-terminal extension of the secreted protein, the translocator domain (or b-domain). This domain is presumed to insert into the outer membrane to form a pore, through which the passenger domain is secreted. No dedicated machinery appears to be required for this step, hence the designation autotransporter.

After transport, the passenger may remain attached to the outer membrane, or it may be released, often through autoproteolytic cleavage. Recently, we demonstrated that this cleavage event can also be mediated in N. meningitidis by a dedicated protease, NalP, which itself is an autotransporter (4). The NalP-released forms differ from the autocatalytically processed forms, which may affect the function of these passengers. The role of differential processing is under investigation. Additionally, the mechanism of autotransporter secretion is still under debate. Recently, we solved the structure of the translocator domain of the autotransporter NalP (5). The structure is consistent with the proposed model for autotransporter translocation, but fails to explain the reported translocation of folded passenger domains, since the channel observed is too narrow. We proposed another mechanism, in which the passenger domain is not transported through the translocator domain, but through a channel formed by Omp85, the protein required for OMP assembly and on which also autotransporters are dependent (6).

Besides the secretion systems described above, we are studying the type III secretion system in Yersinia enterocolitica, which is a one-step mechanism dedicated to the injection of cytotoxic proteins into host cells. In this system, we are mainly interested in the structure and the biogenesis of an outer membrane component, YscC, which is a member of the secretin family and requires a dedicated chaperone for its insertion into the outer membrane (7).

Key references:

  1. Filloux A, Bleves S, van Ulsen P, Tommassen J. 2004. Protein secretion mechanisms in Pseudomonas. pp. 749-791. In JL Ramos (ed) Pseudomonas, Vol. 1. Kluwer Academic/ Plenum Publishers, New York
  2. El Khattabi M, van Gelder W, Bitter W, Tommassen J. 2000. Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone. J. Biol. Chem.275:26885-26891.
  3. de Vrind J, de Groot A, Brouwers GJ, Tommassen J, de Vrind-de Jong E. 2003. Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1. Mol. Microbiol.47:993-1006.
  4. van Ulsen P, van Alphen L, ten Hove J, Fransen F, van der Ley P, Tommassen J. 2003. A Neisserial autotransporter NalP modulating the processing of other autotransporters. Mol. Microbiol. 50:1017-1030.
  5. Oomen CJ, van Ulsen P, van Gelder P, Feijen M, Tommassen J, Gros P. 2004. Structure of the translocator domain of a bacterial autotransporter. EMBO J.23:1257-1266.
  6. Voulhoux R, Bos MP, Geurtsen J, Mols M, Tommassen J. 2003. Role of a highly conserved bacterial protein in outer membrane protein assembly. Science299:262-265.
  7. Burghout P, Beckers F, de Wit E, van Boxtel R, Cornelis GR, Tommassen J, Koster M. 2004. Role of the pilot protein YscW in the biogenesis of the YscC secretin inYersinia enterocolitica. J. Bacteriol. 186:5366-5375.