Assembly of the type II secretion machinery of Erwinia chrysanthemi: Direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL

Citation
B. Py et al., Assembly of the type II secretion machinery of Erwinia chrysanthemi: Direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL, J MOL BIOL, 289(3), 1999, pp. 659-670
Citations number
65
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
00222836 → ACNP
Volume
289
Issue
3
Year of publication
1999
Pages
659 - 670
Database
ISI
SICI code
0022-2836(19990611)289:3<659:AOTTIS>2.0.ZU;2-U
Abstract
Erwinia chrysanthemi secretes, by the type II secretory pathway, a large nu mber of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multi protein complex. Here, we investigated interaction between OutE, the putati ve ATP binding component, and OutL, an inner membrane protein. We showed, b y limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for t he OutE/OutL interaction. Moreover, results from the yeast two-hybrid syste m suggested that OutE and OutL are each able to form homomultimers. The reg ion required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational chan ge in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A m otif in OutE. Our results support the view that interaction occurring on th e cytoplasmic side influences the events occurring in the outer membrane. W e discuss a model in which OutE uses ATP to control the assembly of the typ e II secretion machinery. (C) 1999 Academic Press.