Functional signal peptides bind a soluble N-terminal fragment of SecA and inhibit its ATPase activity.
The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA. The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation. Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA. We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.
Departments of Biochemistry & Molecular Biology and Chemistry, University of Massachusetts, Amherst, Massachusetts 01003-4510, USA.
T L
TL
Triplett
A R
AR
Sgrignoli
F B
FB
Gao
Y B
YB
Yang
P C
PC
Tai
L M
LM
Gierasch
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C085044:LamB signal peptide, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C078167:OMPA outer membrane proteins; D001710:Biotin; D000255:Adenosine Triphosphate; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins