SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third α-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a K d of 2.4 μM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.More than one-third of the proteins synthesized inside the cell must be exported to extracytoplasmic locations to perform their functions. In Escherichia coli, many preproteins are recognized and transported by the Sec transport machinery. This secretory pathway has been extensively studied and several of the key proteins involved have been identified and characterized; however, the mechanisms by which the preprotein interacts with the secretion machinery are not clearly understood.In the cytoplasm, SecB, a chaperone, binds preproteins to keep them in an unfolded state and delivers them to the membrane-associated SecA for post-translational export (1, 2). SecA is a critical component of the Sec transport pathway; it recognizes and binds the preprotein and functions as an ATPase. Moreover, conformational changes resulting from the interaction of SecB with SecA are thought to result in the transfer of the preprotein from the chaperone to the ATPase (3, 4). The membrane proteins, SecG, SecD, and SecF, stabilize and stimulate SecA at the membrane, and as a consequence, SecA can deliver the preprotein through the SecYEG pore (5, 6). Some studies indicate binding of ATP causes the dissociation of SecB from the enzyme, and cycles of ATP hydrolysis (7,8) and conformational changes lead to membrane insertion of the SecA-preprotein complex followed by deinsertion of SecA (9, 10). Meyer et al. (11) One SecA dimerization site is located at its C-terminus (18), which also binds SecY, SecB, and phospholipids (23), and not surprisingly, these a...

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the Signal Peptide Binding Groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53-residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acids 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.

Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported via other pathways. While targeting signals for each route have common overall characteristics, individual signal peptides vary greatly in their amino acid sequences. How do these diverse signals interact specifically with the proteins that comprise the appropriate transport machinery and, at the same time, avoid targeting to an alternate route? The recent publication of the crystal structures of components of the Sec transport machinery now allows a more thorough consideration of the interactions of signal sequences with these components.The general secretory (Sec) pathway in Escherichia coli transports the majority of exported E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, and it serves as a model system for the Sec pathway of the eukaryotic endoplasmic reticulum (ER 1 ). Preproteins that are secreted across the inner membrane through the Sec system contain a hydrophobic, cleavable signal peptide (Figure 1) that interacts posttranslationally with SecA in the cytoplasm (Figure 2a). The SecA -preprotein complex associates with SecYEG at the membrane where the preprotein travels through the SecYEG pore (Figure 2b).Following preprotein translocation, signal peptidase cleaves the signal peptide from the mature protein (Figure 2c). We know that a signal peptide is critical for entrance of a preprotein into this pathway yet how signal sequences are recognized and interact specifically with the transport machinery is the subject of intense study.The lack of primary sequence homology among signal peptides was for some time misleading and the possibility that these peptides were nonetheless endowed with specific recognition elements was not actively considered. Indeed, the "helical hairpin hypothesis" put forth in 1981 (1) emphasized the thermodynamic considerations of moving a hydrophilic protein through a hydrophobic membrane in the absence of specific membrane receptors or transport proteins. More recently, the identification of additional transport routes has required that we rethink the role of the signal peptide. Inner membrane proteins are delivered to SecYEG cotranslationally

We have constructed a series of signal sequence mutants that contain negatively charged amino termini and simplified core regions of varying hydrophobicity levels. This series provides a means of exploring the relative roles of the amino terminus and the hydrophobic core region during transport. The signal peptides with highly hydrophobic core regions support a rapid rate of transport in the presence of a negatively charged amino terminus. We have found that these negatively charged mutants are secreted in a manner similar to the wild-type signal sequence; sodium azide and carbonyl cyanide 3-chlorophenylhydrazone treatments indicate that the negatively charged mutants depend on SecA and the protonmotive force, respectively. These same mutants also demonstrate reduced competition with coexpressed beta-lactamase, reflecting the lower overall affinity for the transport pathway due to the net negative charge at the amino terminus. In addition, the pronounced effects of introducing three negative charges support the conclusion that the two regions function in a concerted manner.

In order to titrate the dependence of individual steps in protein transport on signal peptide hydrophobicity, we have examined a series of mutants which involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide. The core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine. Analysis of this series identified a midpoint for rapid precursor processing between alanine to leucine ratios of 6:4 and 5:5 [Doud et al. (1993): Biochemistry 32:1251-1256]. Examination of precursors that are processed more slowly indicates a lower limit of signal peptide hydrophobicity that permits membrane association and translocation. Analysis of precursors that are processed rapidly defines an intermediate range of hydrophobicity that is optimum; above this level precursors become insensitive to transport inhibitors such as sodium azide and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in parallel with substantial inhibition of beta-lactamase processing. Our data indicate that there is a surprisingly narrow range of signal peptide hydrophobicity which both supports transport of the protein to which it is attached and which does not have such a high affinity for the transport pathway that it disrupts the appropriate balance of other secreted proteins.

Many proteins that are synthesized in the cytoplasm of cells are ultimately found in non-cytoplasmic locations. The correct targeting and transport of proteins must occur across bacterial cell membranes, the endoplasmic reticulum membrane, and those of mitochondria and chloroplasts. One unifying feature among transported proteins in these systems is the requirement for an amino-terminal targeting signal. Although the primary sequence of targeting signals varies substantially, many patterns involving overall properties are shared. A recent surge in the identification of components of the transport apparatus from many different systems has revealed that these are also closely related. In this review we describe some of the key components of different transport systems and highlight these common features.

Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.

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...