Abstract
The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.
Highlights
The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh, and the SecYEG translocon in the bacterial plasma membrane
By preparing ribosome-nascent chain (RNC) in a wheat germ translation system that is devoid of SecA, SecB, and trigger factor (TF) and contains only a trace amount of wheat germ signal recognition particle (SRP), we can examine the association of a bacterial signal sequence with purified bacterial SecA, SecB, SRP, or membranes in the absence of any other bacterial proteins and membranes
Because this approach eliminates any chance of interference with ribosome-associated bacterial components that would be present in a bacterial RNC preparation, interpretation of the results is more straightforward
Summary
The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. We have used photocross-linking and other approaches to characterize the association of signal sequences with SecA in the absence of any competing cofactor binding These experiments strongly suggest that cytosolic SecA is involved in the selection and targeting of nascent translocation substrates to the SecYEG translocon
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