Assignment of the functional loci in the colicin E1 molecule by characterization of its proteolytic fragments.

We obtained two fragments of colicin E1 by limited proteolysis with thermolysin. Both of the fragments have an ionophore-like activity. One of the fragments, Th1 (Mr = 18,000), induces an increase in membrane ion permeability comparable with that with whole colicin E1 when assayed with liposomes prepared from soybean phospholipids. This suggests that Th1 is an active fragment of colicin E1. The size of this fragment is about one-third of that of colicin E1 and it was found that the fragment is derived from the COOH-terminal part of colicin E1. A larger fragment, Th2 (Mr = 39,000), was also obtained on thermolysin treatment of colicin E1, which comprises the two-thirds of colicin E1 from the COOH terminus and has a receptor-binding activity in addition to the same ionophore-like activity of Th1. Th2 has the ability to kill osmotic shocked cells but not intact cells, suggesting that the NH2-terminal portion of colicin E1, which was lost in Th2, is responsible for the transport of the colicin molecule through the outer membrane. Thus, the regions which possess or correlate to the ionophore-like, receptor-binding, and transmembrane activities of colicin E1 were assigned to the COOH-terminal, central, and NH2-terminal parts of the molecule, respectively.