Different modes of SecY-SecA interactions revealed by site-directed in vivo photo-cross-linking - PubMed (original) (raw)

Different modes of SecY-SecA interactions revealed by site-directed in vivo photo-cross-linking

Hiroyuki Mori et al. Proc Natl Acad Sci U S A. 2006.

Abstract

While the SecA ATPase drives protein translocation across the bacterial cytoplasmic membrane by interacting with the SecYEG translocon, molecular details of SecA-SecY interaction remain poorly understood. Here, we studied SecY-SecA interaction by using an in vivo site-directed cross-linking technique developed by Schultz and coworkers [Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. (2002) Proc. Natl. Acad. Sci. USA 99:11020-11024 and Chin, J. W., Schultz, P. G. (2002) ChemBioChem 3:1135-1137]. Benzoyl-phenylalanine introduced into specific SecY positions at the second, fourth, fifth, and sixth cytoplasmic domains allowed UV cross-linking with SecA. Cross-linked products exhibited two distinct electrophoretic mobilities. SecA cross-linking at the most C-terminal cytoplasmic region (C6) was specifically enhanced in the presence of NaN(3), which arrests the ATPase cycle, and this enhancement was canceled by cis placement of some secY mutations that affect SecY-SecA cooperation. In vitro experiments showed directly that SecA approaches C6 when it is engaging in ATP-dependent preprotein translocation. On the basis of these findings, we propose that the C6 tail of SecY interacts with the working form of SecA, whereas C4-C5 loops may offer constitutive SecA-binding sites.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Target SecY residues and SecY–SecA cross-linking conditions. (A) The amino acid residues of SecY are displayed to indicate cytoplasmic (C1–C6), transmembrane, and periplasmic regions (8). The residues individually mutated to amber in this study are indicated by solid circles. The SecY alterations that were introduced additionally into some of the amber mutants are shown by arrows with allele names. (B–D%) Demonstration of in vivo photo-cross-linking between SecY and SecA. Cells expressing the amber 434 mutant of SecY–His6–Myc (lanes 1–8) or WT SecY–His6–Myc (lanes 9 and 10) were grown in the presence of pBPA, treated with 0.08% NaN3 for 5 min, and UV-irradiated for the indicated periods of time (min). Membrane proteins were separated by 7.5% SDS/PAGE and analyzed by anti-SecA (B) and anti-Myc (C) immunoblotting. The cross-linked proportions (%) in the membrane-associated SecA molecules (B) are depicted graphically in D.

Fig. 2.

Fig. 2.

Survey of the SecY cytoplasmic residues for pBPA-mediated cross-linking with SecA. Cells expressing SecY–His6–Myc derivatives having pBPA at the indicated amber positions were treated with 0.08% NaN3 for 5 min and UV-irradiated for 3 min, as indicated. Membrane fractions were analyzed by 7.5% SDS/PAGE and anti-SecA immunoblotting. Cross-liked products are indicated by ∗ for lower mobility product and ∗∗ for higher mobility product.

Fig. 3.

Fig. 3.

Effects of NaN3 on SecY–SecA cross-linking at different SecY positions. (A) Cells expressing SecY–His6–Myc derivatives having pBPA incorporated at the indicated positions were treated with 0.08% NaN3 for 5 min at 37°C when indicated by (NaN3, +) and then UV-irradiated as indicated by (UV, +). Membrane fractions were analyzed by anti-SecA immunoblotting. (B) Averaged cross-linking efficiencies (cross-linked proportions in the membrane-bound SecA, after subtraction of nonirradiated control values) from three independent experiments are shown with error bars. Open columns, without NaN3 treatment; solid columns, after NaN3 treatment.

Fig. 4.

Fig. 4.

Effects of function-related secY alterations on the SecY–SecA cross-linking at position 434. (A) The indicated secY mutations were introduced into SecY–His6–Myc having the amber 434 cross-linking target. Cells expressing the resulting double mutant forms of SecY–His6–Myc were grown in the presence of pBPA and treated with NaN3 as indicated, followed by UV irradiation as indicated. Membranes were analyzed by anti-SecA immunoblotting. (B) Averaged cross-linking efficiencies (cross-linked proportions in the membrane-bound SecA, after subtraction of nonirradiated control values) from three independent experiments are shown with error bars. Open columns, without NaN3 treatment; solid columns, after NaN3 treatment.

Fig. 5.

Fig. 5.

In vitro photo-cross-linking at SecY positions 347 and 434. (A) Effects of concurrent translocation. IMVs (30 ng protein/μl) containing pBPA at indicated positions of SecY were mixed with SecA (5 ng/μl) and with ATP (5 mM) and proOmA (1 ng/μl) as indicated. Samples were incubated at 37°C for 20 min, UV-irradiated for 3 min, and analyzed by anti-SecA immunoblotting. (B) SecY C5 domain interacts with the N-terminal 2/3 of SecA. IMVs (30 ng protein/μl) containing pBPA at SecY positions 347 (lanes 1–4) or 434 (lanes 5–8) were mixed with 5 ng/μl of either SecA or its N68 fragment and incubated at 37°C for 5 min, followed by UV irradiation for 3 min as indicated. Membranes were analyzed by anti-SecA (Upper) and anti-SecY (Lower) immunoblottings. A new cross-linked product observed specifically with the combination of the _amber_-347 IMVs and N68 is indicated by the solid circle.

Fig. 6.

Fig. 6.

Different modes of SecA–SecY binding suggested by the cross-linking results. (A) Location of the SecY residues identified as SecA neighbors in the 3D structure model. E. coli SecY was modeled by using the MOE software (38) on the basis of the structure of Methanococcus jammaschii SecYEβ (2). The pseudosymmetrical halves of SecY (ribbon representation) are shown in red for TM1–TM5 and blue for TM6–TM10. SecE and Secβ are shown for reference purpose in pale blue and green, respectively, as unaltered M. jammaschii subunits. Side chains of the indicated residues, identified as SecA neighbors, are space-filled. (B) A schematic representation of different modes of SecA–SecY binding suggested by the present results. The different electrophoretic mobilities of the cross-linked products are assumed to result from the cross-linked positions on SecA, within the N-terminal 2/3 (N68) region for the lower mobility and within the C-terminal 1/3 domain for the higher mobility. The cross-linking via the C6 residues is enhanced by ongoing translocation reaction. The present study revealed three different modes (i, ii, and iii) of interaction between SecA and SecY.

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