Protein secretion is an essential cellular process. Signal sequences, RNA-containing targeting factors (Signal Recognition Particle, SRP), and a translocation machinery that contains a heterotrimeric complex o membrane proteins have been discovered in all three domains of life. In Escherichia coli, signal sequences are found on proteins destined for the inner or outer membranes, the periplasmic space located between these lipid bilayers, and on proteins that are secreted into the growth media. The prokaryotic SEP (pSRP) containsa protein, Ffh and 4.5S RNA, and the heterotrimeric membrane protein complex is comprised of SecY (PrlA), SecE (PrlG), and SecG (PrlH). Secretory components that are unique to bacteria include the secretion specific chaperone, SecB, and a translocation ATPase, SecA (PrlD). The prl alleles of the various sec genes are suppressor mutations that restore the secretion o: preproteins with defective signal sequences. We have shown that PrlA and PrlG act by abolishing a signal sequence proofreading function, and we will test our hypothesis that signal sequences interact directly with transmembrane helix 7 of SecY. In vivo crosslinking of SecY and SecE by disulfide bonding, and depletion of Ffh cause similar phenotypes. We will test whether this indicates a more fundamental role for pSRP. We will also explore the possibility that a novel mutation,prlFl, affects a targeting activity of SecYEG for inner membrane proteins. We have discovered partially overlapping signal transduction pathways (Cpx and aE) that control the synthesis of periplasmic protein folding factors and a protease. We will continue our search for additiona factors and we will investigate how these molecules function in outer membrane biogenesis. In addition, we will determinethe mechanism by which the histidinekinase CpxA senses envelope stress, and we will test our prediction that Cpx is the master regulator of the """"""""attached"""""""" phase during urinary tract infection. Results o: these studies may suggest new ways to treat or prevent certain types of bacterial infections.
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