Marín E, Bodelón G & Fernández LA.
Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-domain that is inserted in the outer membrane (OM) and is essential for translocation of a N-terminal passenger domain to the extracellular milieu.
Several mechanisms have been proposed for ATs secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the 
-barrel or by oligomerization of C-domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam-complex. In this work we have investigated ATs secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-domains selected from ATs of 
, , 
and 
class of proteobacteria.
Our results indicate that C-domains having an N-terminal -helix and a -barrel constitute functional transport units for translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analysis show that multimerization is not a conserved feature in AT C-domains. Further, we demonstrate that deletion of the conserved -helix severely impairs -barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that AT -barrel without its -helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for understanding AT secretion are discussed