RESUMO
SecA signal peptide interaction is critical for initiating protein translocation in the bacterial Sec-dependent pathway. Here, we have utilized the recent nuclear magnetic resonance (NMR) and Förster resonance energy transfer studies that mapped the location of the SecA signal peptide-binding site to design and isolate signal peptide-binding-defective secA mutants. Biochemical characterization of the mutant SecA proteins showed that Ser226, Val310, Ile789, Glu806, and Phe808 are important for signal peptide binding. A genetic system utilizing alkaline phosphatase secretion driven by different signal peptides was employed to demonstrate that both the PhoA and LamB signal peptides appear to recognize a common set of residues at the SecA signal peptide-binding site. A similar system containing either SecA-dependent or signal recognition particle (SRP)-dependent signal peptides along with the prlA suppressor mutation that is defective in signal peptide proofreading activity were employed to distinguish between SecA residues that are utilized more exclusively for signal peptide recognition or those that also participate in the proofreading and translocation functions of SecA. Collectively, our data allowed us to propose a model for the location of the SecA signal peptide-binding site that is more consistent with recent structural insights into this protein translocation system.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Espectroscopia de Ressonância Magnética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutação , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Canais de Translocação SEC , Proteínas SecARESUMO
Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.