These sites were identified as contact sites between SecA and its precursor ligands by site-directed spin labeling and electron paramagnetic resonance spectroscopy (3)

These sites were identified as contact sites between SecA and its precursor ligands by site-directed spin labeling and electron paramagnetic resonance spectroscopy (3). others, such as OmpA, are inserted into the outer membrane. GSK-3787 The path across the cytoplasmic membrane barrier is provided by a translocation GSK-3787 channel comprising a heterotrimeric complex, SecY, SecE, and SecG (SecYEG). The energy for the movement is supplied by protonmotive force and the hydrolysis of ATP by SecA, which is a peripheral component of the membrane-associated translocon. The Sec system can export polypeptides only if they are devoid of stable tertiary structure. SecB, a small cytosolic chaperone, acts to capture precursors before they acquire stable structure and introduces them into the secretory pathway by delivering Rabbit Polyclonal to SLC27A4 them to SecA. SecA can also bind precursors directly, as evidenced by the viability of strains ofE. colithat GSK-3787 lack SecB. However, efficiency of export GSK-3787 is drastically reduced for many proteins (12). Crystal structures of both SecB and SecA have been solved. SecB is a tetramer (monomer, 17 kDa) organized as a dimer of dimers (6,33). There are six dimeric forms of SecA (2,10,19,26,30,34) that differ greatly in the contacts that stabilize the dimeric interface. However, the structures of the protomers are all closely related and display only two different conformations, an open state (17) and a closed state (10). In free solution, SecA exists in a dynamic equilibrium between monomer and dimer characterized by an equilibrium constant of 0.1 M to 1 1 M, depending on the ionic strength and temperature of the solution (31). When SecA interacts with SecB, two protomers must be bound to SecB for the complex to be active, as assessed in vitro (23). In this study, we asked why two protomers of SecA are required. We examined the translocation into inverted cytoplasmic membrane vesicles of two natural ligands, one a soluble periplasmic protein, galactose-binding protein, and the other an outer membrane protein, OmpA. We conclude that two protomers of SecA in the complex are required to achieve maximal coupling efficiency between ATP hydrolysis and translocation. == MATERIALS AND METHODS == == Materials. == [-32P]ATP was purchased from either PerkinElmer (Boston, MA) or GE Healthcare (Pittsburgh, PA), [35S]methionine from PerkinElmer, precoated polyethyleneimine cellulose thin-layer chromatography plates from Merck KGaA (Darmstadt, Germany), trypsin from Millipore Corporation (Freehold, NJ), bovine pancreatic trypsin inhibitor (aprotinin) and DNase I from Sigma-Aldrich (St. Louis, MO), NAP10 and HiTrap Blue HP columns from GE Healthcare Bio-sciences AB (Uppsala, Sweden),N-[(2-pyridyldithio)ethyl]-4-azidosalicylamide (AET) from Toronto Research Chemicals Inc. (Toronto, Canada), Precision Plus protein standards (all blue) from Bio-Rad (Hercules, CA), andStaphylococcus aureusmicrococcal nuclease from Worthington (Lakewood, NJ). == Protein purification. == SecA, SecAC4, SecAdN7, SecAdN10, SecB, and SecBL75Q were purified from strains ofE. coliharboring plasmids that express the proteins as previously described (23,24), except that cells expressing wild-type SecA and those expressing SecAdN10 were suspended at 2 g (wet weight) of cell pellet per ml of buffer and disrupted using a French press at 8,000 lb/in2. The P11 column was omitted from the SecAdN10 purification. For cross-linking experiments, SecAC4S350C and SecAC4I641C were purified from strains harboring plasmids derived from plasmid pT7secAC4, which carries a gene for SecA in which the four native cysteine codons are replaced by serine codons GSK-3787 (22). For each SecA species, a single cysteine was introduced at the site of interest by site-directed mutagenesis (QuikChange; Stratagene). Proteins were purified as described previously (23) with minor changes. Micrococcal nuclease (314 units/ml [final]) was included along with DNase I (5,000 devices/ml [final]) to degrade nucleic acids and decrease the viscosity of the lysate. SecAC4S350C was purified by chromatography on a HiTrap QAE column followed by a HiTrap Blue HP column instead of P11. For purification of SecAC4I641C, only a HiTrap Blue HP column was used. Precursor galactose-binding protein was purified as explained previously (28). The precursor of OmpA labeled with [35S]methionine was produced from.