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
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
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.