Role of SecA and SecY in protein export as revealed by studies of TonA assembly into the outer membrane of Escherichia coli.

The growth of secAts or secYts mutants at the restrictive temperature has been shown to inhibit the export of many outer membrane proteins. We report here that in two secAts strains the rate of incorporation of newly synthesized protein into both inner and outer membrane fractions decreased by about 70% at the restrictive temperature. The export of the outer membrane protein TonA was used as a model system in which to study the effects of SecA or SecY inactivation. pre-TonA that accumulated at the restrictive temperature was found to co-sediment with the outer membrane fraction. However, the precursor was sensitive to protease and did not float up a sucrose gradient with the membrane fractions. It was therefore concluded that pre-TonA was not integrated into the outer membrane fraction but probably accumulated in the cytoplasm. Studies on the rate of processing of pre-TonA, pulse-labelled at the restrictive temperature then chased at the permissive temperature, revealed differences between secA and secY mutants. In the secAts mutant the great majority of cytoplasmic pre-TonA was not apparently processed to the mature form, whereas in the secYts mutant significant amounts of precursors were rapidly chased into mature TonA, which appeared in the outer membrane. These results suggest that SecA and SecY may act sequentially in the export of proteins to the outer membrane. In particular these data indicate that SecA is required to maintain pre-TonA in a translocationally competent form prior to interaction with the SecY export site.