Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

Abstract

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. † This research was supported in part by National Institutes of Health Grant GM37639 (to D.A.K. Protein transport across, or integration into, biological membranes is a vital cellular process (1-3). Components of the Sec translocon, the membrane pore through which presecretory proteins (or membrane proteins) achieve membrane translocation (or integration), are the most conserved transport constituents throughout the three kingdoms of life (4).In Escherichia coli, the essential components of the translocase (5) include the membraneassociated form of SecA (6, 7) and the polytopic membrane proteins SecY, SecE (homologues of the mammalian Sec61α, Sec61γ, and the yeast ER 1 Sec61p, Sss1p, respectively), and SecG (8, 9); the latter three proteins form a stable trimeric SecYEG complex (10). SecA is an ATPase that powers the membrane translocation of hydrophilic polypeptides by coupling ATP hydrolysis with protein movement via concomitant SecA membrane insertion and deinsertion cycles (11,12). SecY protein has 10 transmembrane (TM1-TM10), six cytosolic (C1-C6), and five periplasmic (P1-P5) domains (13), and it forms the core of the passageway for the translocating polypeptide chain (14 (35,36). Suppressor analysis of prlA mutations using dysfunctional LamB signal peptides revealed that the suppressor mutations clustered in distinct regions, and TM7 of SecY was proposed to function in signal sequence recognition (37). Yet no investigation has focused on the direct interaction between a signal peptide and the E. coli translocon. Consequently, it remains unclear whether the nascent polypeptide chain is merely translocating in close proximity to SecY, SecY performs simply a proofreading function (37), or SecY is more intimately involved in the specific recognition of the signal peptide. Wang et al. Page 2Biochemistry. Author manuscript; available in PMC 2011 April 29. NIH-PA Author ManuscriptNIH-PA Author Manuscr...

Probing the Affinity of SecA for Signal Peptide in Different Environments

“…In addition, the presence of nucleotide reduces further the affinity of lipid-bound SecA for the signal peptide, consistent with ADP binding inhibiting the endothermic conformational transition of SecA (30). In the context of translocation, this may help to ensure that one consequence of SecA-signal peptide membrane insertion is release of the signal peptide for interaction with SecY (13).…”

“…microscopy, that oligomers of SecYEG form protein-conducting channels in lipid bilayers that are sufficiently wide to accommodate part of SecA with the preprotein, yet the recent Xray structure of SecYEG (12) revealed that the channel is too small to allow insertion of SecA; rather, the SecYEG pore is large enough only for preprotein incorporation, consistent with signal peptide recognition by SecYEG (13).…”

Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

“…7,20 The photoactive analogue of this peptide, in which phenylalanine was substituted by ρ-benzoylphenylalanine (Bpa), MKQSTIALALLPLL(Bpa) TPVTKAC-NH 2 , was chemically synthesized and biotinylated by thiol linkage with cysteine as described by Wang et al 19 The activity of the peptide was confirmed by measuring the extent of stimulation of SecA-lipid ATPase activity as described previously. 5,7…”

Coarse Grained Protein−Lipid Model with Application to Lipoprotein Particles

“…The associated cholesterol is then esterified by lecithin cholesterol acyl transferase and the HDL particles undergo a structural change, going from discoidal to spherical in shape. 19 Nanodiscs are protein-lipid particles which are engineered to mimic nascent discoidal HDL particles. They are produced in order to create a platform in which to embed and solubilize membrane proteins in a native lipid environment.…”