- We know very little about how the cellular environment affects protein folding mechanisms. Here, we focus on one unique aspect of that environment that is difficult to recapitulate in the test tube: the effect of a folding vector. When protein folding is initiated at one end of the polypeptide chain, folding starts from a much smaller ensemble of conformations than during refolding of a full-length polypeptide chain. But to what extent can vectorial folding affect protein folding kinetics and the conformations of folding intermediates? We focus on recent studies of autotransporter proteins, the largest class of virulence proteins from pathogenic Gram-negative bacteria. Autotransporter proteins are secreted across the bacterial inner membrane from N→C-terminus, which, like refolding in vitro, retards folding. But in contrast, upon C→N-terminal secretion across the outer membrane autotransporter folding proceeds orders of magnitude faster. The potential impact of vectorial folding on the folding mechanisms of other proteins is also discussed.