Phospholipid-induced Monomerization and Signal-peptide-induced Oligomerization of SecA

Benach, J.; Chou, Yi-Te; Fak, John; Itkin, Anna; Nicolae, Daita D.; Smith, Paul; Wittrock, Guenther; Floyd, Daniel L.; Golsaz, Cyrus M.; Gierasch, Lila M.; Hunt, J.F.

doi:10.1074/jbc.m205992200

Cited by 99 publications

(123 citation statements)

References 68 publications

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“…A comparison of our experimental setup with that of other studies, in which water-soluble lipids are found to promote SecA monomers but signal peptide induces dimers (26) or lipid induces monomers and signal peptide maintains that state (27), reveals a few key elements become apparent. Monomeric SecA is found in the presence of the signal peptide when the lipid:SecA ratio is at least 950:1 and with the signal peptide concentration lower than 100 μM (this study and ref 27).…”

Section: Discussionmentioning

confidence: 93%

“…According to some experiments, SecA exists as a monomer with some dimer present when bound to SecYEG (25). Benach et al (26) have observed that SecA is a dimer in solution with or without the signal peptide bound, while the protein monomerizes when bound to lipids and redimerizes with addition of peptide. Fluorescence studies also show that E. coli phospholipids monomerize SecA, but suggest that it remains monomeric upon peptide addition (27).…”

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confidence: 99%

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Probing the Affinity of SecA for Signal Peptide in Different Environments

2005

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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...

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Section: Discussionmentioning

confidence: 93%

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confidence: 99%

Probing the Affinity of SecA for Signal Peptide in Different Environments

2005

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“…For example, distortion of the lateral pressure profile, a mechanism that has been attributed to general anesthetics (29), may lead to increased lateral pressure, which in turn may physically inhibit transmembrane channels (30). Additionally, peptides corresponding to the Escherichia coli LamB signal peptide have been shown to affect the oligomerization of integral transmembrane proteins (31). Such surface activity is consistent with the SHP-1 site of action being the plasma membrane, as suggested by killing assays performed at 3°C (Fig.…”

Section: Discussionmentioning

confidence: 99%

The Plasma Membrane of Bloodstream-form African Trypanosomes Confers Susceptibility and Specificity to Killing by Hydrophobic Peptides

Harrington

Widener

Stephens

et al. 2010

Journal of Biological Chemistry

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Trypanosoma brucei is the causative agent of both a veterinary wasting disease and human African trypanosomiasis, or sleeping sickness. The cell membrane of the developmental stage found within the mammalian host, the bloodstream form (BSF), is highly dynamic, exhibiting rapid rates of endocytosis and lateral flow of glycosylphosphatidylinositol-anchored proteins. Here, we show that the cell membrane of these organisms is a target for killing by small hydrophobic peptides that increase the rigidity of lipid bilayers. Specifically, we have derived trypanocidal peptides that are based upon the hydrophobic N-terminal signal sequences of human apolipoproteins. These peptides selectively partitioned into the plasma membrane of BSF trypanosomes, resulting in an increase in the rigidity of the bilayer, dramatic changes in cell motility, and subsequent cell death. No killing of the developmental stage found within the insect midgut, the procyclic form, was observed. Additionally, the peptides exhibited no toxicity toward mammalian cell lines and did not induce hemolysis. Studies with model liposomes indicated that bilayer fluidity dictates the susceptibility of membranes to manipulation by hydrophobic peptides. We suggest that the composition of the BSF trypanosome cell membrane confers a high degree of fluidity and unique susceptibility to killing by hydrophobic peptides and is therefore a target for the development of trypanocidal drugs.

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“…In addition, the study of NBF-I and NBF-II interaction should be undertaken as well, since it controls the accessibility of the high affinity nucleotide-binding site of SecA and has been proposed to regulate multiple ATP turnover events in concert with C-domain modulation (15,45). Finally, with the recent finding of a dynamic SecA monomer-dimer equilibrium during protein translocation conditions (46)(47)(48), FRET studies could also be used to address when and how SecA monomerizes or whether an alternative dimer forms during this process. This basic approach should lend additional insight at the structural level to SecA function and mechanism.…”

Section: Discussionmentioning

confidence: 99%