Structure and Electrophysiological Properties of the YscC Secretin from the Type III Secretion System of Yersinia enterocolitica

Abstract

YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.The outer membrane of gram-negative bacteria not only protects the cell against harmful compounds in the extracellular environment, such as antibiotics, detergents, and digestive enzymes, but also forms a barrier to the secretion of proteins and uptake of nutrients. The passage of small hydrophilic molecules across the outer membrane is facilitated by the presence of a large number of proteinaceous channels, formed by general and substrate-specific porins and TonB-dependent receptors (31, 41). Gram-negative bacteria have evolved several specialized pathways for the secretion of proteins. These pathways all require one or more integral outer membrane proteins, which in the cases of the type II and III protein secretion systems are related and belong to the family of secretins (19).

Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

“…Interestingly, oligomeric secretins such as XcpQ from Pseudomonas aeruginosa (34) and YscC from Yersinia enterocolitica (35) form channels with very large conductances (ϳ3-10 nS), which are comparable with that of the ⌬plug PapC mutant. EM studies revealed that the monomers organize as ring-like structures forming a central pore (34,35). These observations are consistent with the notion that a fairly large pore is required for the translocation of folded substrates by secretion systems, either as a central conduit within a single subunit (as in PapC) or as the center of a ring of monomers (as in YscC and XcpQ).…”

Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

“…In contrast, the T3 secretin complex of Y. enterocolitica was shown to be a 13-mer ( Fig. 1b; [47]). Given their mass, the two minor peaks on this histogram arise from the aggregation of two and three complexes, respectively.…”

“…These are present in the bacterial outer membrane as homo-oligomers. STEM has been employed to define the oligomerisation state of two; the T2 secretin complex of K. oxy toca formed by the protein PulD and the corresponding T3 complex of Y. enteroco litica formed by the protein YscC [47] [48]. In the more extensive study made on the K. oxytoca system [48][49], STEM mass measurements indicated the presence of twelve monomeric subunits in the intact PulD complex and in its trypsin-resistant fragment ( Fig.…”

The Extra-Membranous Domains of the Competence Protein HofQ Show DNA Binding, Flexibility and a Shared Fold with Type I KH Domains

“…17,19 Secretins are large homo-oligomeric assemblies built up of 50-70 kDa subunits, with 12-14 subunits forming a ring structure of approximately 100-150 Å in diameter. 20,21,22,23,24,25,26,27,28 They comprise a superfamily 29,30 and form components of several distinct secretion systems in the outer membrane, including the type-two secretion system (TIISS), 31,32 type-three secretion system (TIIISS) 26 and Type-IV pilus biogenesis system. 30,33,34 All secretins consist of two major regions.…”