Summary auto-generated
This study characterized the membrane topology of four proteins (OutC, OutD, OutE, and OutF) from the general secretion pathway apparatus of Erwinia carotovora subsp. carotovora, a Gram-negative bacterium that secretes pectinase and cellulase enzymes. Researchers constructed fusions between out genes and blaM (a β-lactamase topology probe) to determine protein localization in both E. coli and the native host. OutC was confirmed as a bitopic cytoplasmic membrane protein with a single transmembrane domain and a large periplasmic domain. OutF was identified as a polytopic membrane protein with three transmembrane regions connecting a large N-terminal cytoplasmic domain, a smaller periplasmic loop, and a large cytoplasmic domain. Additional analysis confirmed OutD localizes to the outer membrane and OutE to the cytoplasm. These topological findings suggest the Out proteins possess multiple domains capable of protein-protein interactions necessary for facilitating secretion of enzyme intermediates across the outer membrane to the external environment. The detailed membrane organization provides insights into how this conserved secretion system functions across diverse bacterial species.
Key findings
- OutC adopts a type II bitopic conformation in the cytoplasmic membrane with one transmembrane domain and a periplasmic C-terminal domain
- OutF is a polytopic inner-membrane protein containing three transmembrane domains connecting two large cytoplasmic regions and one small periplasmic loop
- OutD localizes to the outer membrane while OutE localizes to the cytoplasm, confirmed by β-lactamase fusion analysis
- Multiple Out proteins possess domains in both the cytoplasm and periplasm, providing potential sites for protein-protein interactions essential for the secretion pathway
- The detailed topology maps support computer-predicted structures and reveal how these proteins may interact to form a functional secretion complex
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Abstract
The out gene cluster of Erwinia carotovora subsp. carotovora (Ecc) encodes the proteins of the type II or general secretory pathway (GSP) apparatus which is required for secretion of pectinase and cellulase. In this study, fusions between Ecc out genes and the topology probe blaM were constructed. The ability of Out protein domains to export BlaM across the cytoplasmic membrane in both Escherichia coli and the cognate host was utilized to confirm the computer-predicted cytoplasmic membrane topology of OutC and OutF, When outC was fused to blaM, the resulting phenotype suggested that the majority of OutC is targeted to the periplasm, typical of a type II bitopic conformation in the cytoplasmic membrane. In contrast, for the outF gene product, three transmembrane regions were identified which connect a large N-terminal cytoplasmic domain, a smaller periplasmic domain, and a large cytoplasmic loop. Fusions between blaM and outD and outE were used to further substantiate the locations of these gene products in the outer membrane and the cytoplasm respectively. The data derived suggest that a number of the Out apparatus components possess domains in the cytoplasm and/or the periplasm with potential for protein-protein interactions which facilitate the secretion of periplasmic enzyme intermediates across the outer membrane to the external milieu.