The Bacterial SRP Receptor, SecA and the ... - Wiley Online Library

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© 2011 John Wiley & Sons A/S doi:10.1111/j.1600-0854.2011.01167.x

The Bacterial SRP Receptor, SecA and the Ribosome Use Overlapping Binding Sites on the SecY Translocon Patrick Kuhn1,2 , Benjamin Weiche1 , Lukas Sturm1 , Erik Sommer3 , Friedel Drepper2,4 , Bettina Warscheid2,4 , Victor Sourjik3 and Hans-Georg Koch1,∗ 1 Institut

¨ Biochemie und Molekularbiologie, ZBMZ, fur Stefan-Meier-Str. 17, D-79104 Freiburg, Germany 2 Fakultat ¨ fur ¨ ¨ Biologie, Schanzlestr. 1, D-79104 Freiburg, Germany 3 Zentrum fur ¨ ¨ Molekulare Biologie der Universitat Heidelberg, DKFZ-ZMBH-Alliance, 69120 Heidelberg, Germany 4 Centre for Biological Signalling Studies (Bioss), ¨ Freiburg, 79104 Freiburg, Albert-Ludwigs-Universitat Germany *Corresponding author: Hans-Georg Koch, [email protected] Signal recognition particle (SRP)-dependent protein targeting is a universally conserved process that delivers proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum membrane in eukaryotes. Crucial during targeting is the transfer of the ribosome-nascent chain complex (RNC) from SRP to the Sec translocon. In eukaryotes, this step is co-ordinated by the SRβ subunit of the SRP receptor (SR), which probably senses a vacant translocon by direct interaction with the translocon. Bacteria lack the SRβ subunit and how they co-ordinate RNC transfer is unknown. By site-directed cross-linking and fluorescence resonance energy transfer (FRET) analyses, we show that FtsY, the bacterial SRα homologue, binds to the exposed C4/C5 loops of SecY, the central component of the bacterial Sec translocon. The same loops serve also as binding sites for SecA and the ribosome. The FtsY–SecY interaction involves at least the A domain of FtsY, which attributes an important function to this so far ill-defined domain. Binding of FtsY to SecY residues, which are also used by SecA and the ribosome, probably allows FtsY to sense an available translocon and to align the incoming SRP–RNC with the protein conducting channel. Thus, the Escherichia coli FtsY encompasses the functions of both the eukaryotic SRα and SRβ subunits in one single protein.

bacteria to humans (1). SRP recognizes its cargo proteins already early during translation (2,3) and targets the ribosome-nascent chain complexes (RNCs) to the membrane-bound SRP receptor (SR) (1). Upon binding of the SRP–RNC complex to SR, the translocon-binding site of the ribosome is exposed allowing the docking of the RNC onto the Sec translocon (4,5). This aligns the ribosomal tunnel with the translocon channel and allows the passage of the newly synthesized protein into the lumen/membrane of the endoplasmic reticulum (ER) in eukaryotes or into the cytoplasmic membrane in bacteria. Through subtle conformational changes, GTP binding and hydrolysis by SRP and SR are modulated, which implement proofreading steps that strongly enhance the fidelity of the targeting reaction (6–8). Although both eukaryotic and bacterial cells share these principles of SRP-dependent targeting, there are differences in substrate specificity and subunit composition. SRP in eukaryotes recognizes both secretory and membrane proteins, while in bacteria the SRP pathway is mainly used for inner membrane proteins (1,9). Bacterial secretory proteins, on the other hand, are targeted primarily via the post-translational SecA-dependent pathway to the Sec translocon (9). In general, the bacterial SRP and SR represent simplified versions of their mammalian counterparts (1). Substrates are recognized in bacteria by the Ffh subunit, which together with the 4.5S RNA forms a minimal SRP. In eukaryotes, the Ffh homologue SRP54 is required for substrate recognition and is also bound to an RNA molecule, the 7SL RNA. However, the eukaryotic SRP contains five additional subunits and can be functionally divided into the S domain, which is responsible for substrate recognition, and the Alu domain, which is involved in slowing down translation during targeting (10). Owing to its simplicity, this latter function is not present in the bacterial SRP, although bacteria might have Alu domainindependent mechanisms of translation regulation (11,12).

Received 26 July 2010, revised and accepted for publication 19 January 2011, uncorrected manuscript published online 21 January 2011, published online 25 February 2011

The SR in bacteria is also simpler than the eukaryotic SR. It consists of only the FtsY subunit (13), which is homologous to the eukaryotic SRα and essential for recruiting the SRP–RNC complex to the membrane. The eukaryotic SRα is tethered to the membrane via its interaction with the membrane-integral SRβ subunit (14,15), which is not present in bacteria. Instead, membrane binding of most bacterial FtsY is achieved via two lipid-binding helices, which are N-terminally fused to the conserved NG-domain of FtsY (16–19).

The signal recognition particle (SRP) pathway is an essential protein targeting mechanism that is conserved from

In contrast to its GTPase domain, the transmembrane domain of SRβ is not essential for cotranslational targeting (15), suggesting that tethering SRα to the membrane

Key words: cotranslational protein targeting, ribosome, SecA, Sec translocon, signal recognition particle, SRP receptor, trigger factor

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is not the primary function of SRβ. Instead, several reports indicate that SRβ is crucial for co-ordinating the transfer of the SRP–RNC complex to the Sec translocon. Proximity between SRβ and the Sec translocon has been observed (20) and genetic and biochemical data strongly suggest that the eukaryotic SR interacts with the β-subunit of the Sec translocon (21). This interaction could facilitate the recognition of unoccupied translocons, thereby allowing the transfer of the RNCs from the SRP–SR complex to the Sec translocon. How bacteria co-ordinate this transfer in the absence of SRβ is unknown; in particular, because also SecG, the third subunit of the bacterial Sec translocon, shows no similarity to the β-subunit of the eukaryotic translocon (22). Thus, bacteria need to employ a different mechanism to recognize empty translocons and to align the RNCs onto the Sec translocon. Previous in vitro data have shown that FtsY can interact at least transiently with the bacterial translocon (23,24), however, the mode of binding and the exact contact sites were unknown. Here, we found that FtsY binds at least via its A domain to the SecY translocon. FtsY binds to the C-terminal loops of SecY and occupies the same binding sites on the Sec translocon as the ribosome and SecA. This direct binding of FtsY to the SecY translocon probably allows bacteria to achieve the recognition of an empty translocon and the subsequent positioning of the incoming RNC in a single step.

The yellow fluorescent protein (YFP) was fused to the C-terminus of Ffh and served as an acceptor fluorophore. Both fluorescently labelled proteins were functional in vivo, because they were able to complement conditional FtsY/Ffh mutant strains (Figure S1). The expression level of FtsY-Cer was comparable to the expression of endogenous FtsY (Figure S1), while the level of Ffh-YFP expression was higher than the endogenous Ffh expression, which is expected due to the low copy number of Ffh in E. coli [approximately 100 molecules/cell; (27)]. The functionality of FtsY-Cer and Ffh-YFP was further verified by testing their GTPase activity. The interaction between FtsY and SRP leads to their reciprocal GTPase stimulation (28) and the GTPase activation of the fluorescently labelled protein was comparable to the GTPase activation of the non-labelled proteins (Figure S1), which further shows that the fluorescent tag did not significantly influence FtsY–Ffh interaction. When both FtsY-Cer and Ffh-YFP were coexpressed in E. coli, FtsY-Cer was almost exclusively membrane localized (Figure 1A), as

Results and Discussion Determining the FtsY–SecY interaction by in vivo FRET analyses Based on in vitro studies, we had previously postulated that FtsY is able to directly interact with the SecY translocon (23,24). For further defining the interaction between both proteins in living Escherichia coli cells, we employed an in vivo fluorescence resonance energy transfer (FRET) approach, which allows the non-invasive detection of intracellular protein–protein interactions. If two labelled proteins form a complex, the energy transfer from an excited donor fluorophore to an acceptor fluorophore leads to a quenching of the donor fluorescence. The specificity of the FRET assay is ensured by the steep dependence of energy transfer efficiency on the distance between the fluorophores, with essentially no FRET being observed at distances above 10 nm for green fluorescent protein (GFP)-based FRET pairs. The FRET efficiency can be detected as an increase in donor fluorescence after photobleaching the acceptor (25). For determining whether the in vivo FRET approach was suitable for detecting protein–protein interactions during cotranslational targeting, we first analysed the established interaction between FtsY and SRP in living E. coli cells. As a donor, we fused the monomeric fluorescent protein Cerulean (Cer) (26), a version of the cyan fluorescent protein, to the C-terminus of FtsY. 564

Figure 1: FRET analyses show that FtsY binds to the SecY translocon in living E. coli cells. A) The correct localization of the fluorescently tagged proteins was verified by fluorescence microscopy as described in Materials and Methods. In some cells expressing YFP-SecY or SecY-YFP, SecY was not evenly distributed but showed some spotted appearance (arrows). Ffh-YFP was accumulated in the septal area in some cells. The white scale bar corresponds to 1 μm. B) In vivo FRET analyses of the indicated donor and acceptor pairs. Proteins were coexpressed in E. coli and the energy transfer was determined by measuring donor fluorescence before and after bleaching of the acceptor fluorophore YFP. FRET signals are the results from at least two independent experiments.

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FtsY–SecY Translocon Interaction

described previously (19), while Ffh-YFP was found both in the cytosol and at the membrane, with an accumulation in the septal area in some cells (Figure 1A). In cells coexpressing FtsY-Cer and Ffh-YFP, we observed efficient energy transfer in vivo (Figure 1B), confirming that this approach is suitable for detecting protein–protein interactions during cotranslational targeting. It has to be noted that because of a relatively high background arising from cellular autofluorescence and excitation light scattering by cells, the apparent FRET efficiency determined using acceptor photobleaching in vivo is substantially lower than the theoretical efficiency of energy transfer for the same pair (see Materials and Methods). Therefore, apparent FRET efficiency above 0.5% is typically taken as a criterion of significant interactions in this setup, and FRET efficiency of 2.5%, as observed for FtsY and Ffh, represents a strong interaction (25). To exclude false-positive FRET due to heterodimerization of both fluorophores, we coexpressed Ffh-YFP with just the donor fluorophore Cerulean (Figure 1B). Here, we did not observe FRET, which excludes that unspecific interactions between the two fluorophores contribute to the FRET efficiencies. YFP was then fused to either the N-terminus (YFP-SecY) or C-terminus (SecY-YFP) of SecY and the fusion proteins were coexpressed with FtsY-Cer. We observed significant FRET efficiency between FtsY-Cer and SecY-YFP (Figure 1B), while the FRET signal with YFP-SecY was slightly below the threshold level of 0.5%. A FRET signal below the threshold level was also observed for the SecY-Cer/Ffh-YFP pair (Figure 1B), which was used as an additional negative control, because SRP is not expected to interact directly with the SecY translocon. YFP-SecY and SecY-YFP were expressed at comparable amounts

and their expression level was only slightly higher than the endogenous SecY concentration (Figure S2). Both constructs localized to the membrane (Figure 1A), but were not evenly distributed within the membrane, which is in agreement with previously published data (29). The activity of N- and C-terminally fused SecY derivatives was analysed in a complementation assay using the temperature-sensitive SecY mutant strain SecY24 (30). These analyses showed that although both constructs were able to complement this conditional SecY mutant, the YFP–SecY construct was less active than wild-type SecY (Figure S2). We did not detect the free fluorophore in cells expressing YFP-SecY or SecY-YFP (Figure S2), indicating that the complementation is not because of regenerating SecY by cleaving off the fluorescent tag. In summary, these data substantiate our previous in vitro observations and show that FtsY and SecY interact in living E. coli cells. However, it is important to note that the YFP-SecY derivative was not fully active (Figure S2), and therefore the differences in FRET efficiency between FtsY-Cer/SecY-YFP and FtsY-Cer/YFPSecY do not necessarily mean that FtsY is only interacting with the C-terminus of SecY. For obtaining a more detailed topological information about the FtsY–SecY interaction, we therefore employed a different in vivo approach.

FtsY binds to the fourth and fifth cytosolic loops of SecY For determining the binding site for FtsY on the SecY translocon, we used an in vivo site-directed cross-linking approach with the phenylalanine derivative p-benzoylL-phenylalanine (pBpA; 31). Amber-stop codons were first incorporated at different positions within all six membrane-exposed cytosolic loops of SecY (Table 1),

Table 1: Mapping the SecY interactions with cytosolic ligands by in vivo cross-linking Mutant phenotype Position 11 111 179 238 240 250 255 259 347 351 356 357 363 429

Cross-links to

Loop

Corresponding mutant

Protein integration/ SRP pathway

Protein translocation/ SecA pathway

FtsY

SecA

L23

Others

C1 C2 C3 C4 C4 C4 C4 C4 C5 C5 C5 C5 C5 C6

Not known Not known Not known SecY238 (E→K) SecY24 (G→D) Not known Not known Not known Not known SecY351 (A→T) Not known SecY39 (R→H) SecY40 (A→T) SecY205 (Y→D)

Not known Not known Not known Weakly impaired Impaired Not known Not known Not known Not known Weakly impaired Not known Impaired Impaired Active

Not known Not known Not known Weakly impaired Impaired Not known Not known Not known Not known Impaired Not known Impaired Active Impaired

− − − − − − + + − − − + + −

− + − − − + + (+) + (+) − + − +

− − − − − − − − − − − + − −

— SecG SecG — SecE — SecG SecG — — — — — —

Positions correspond to the amino acid numbering of the E. coli SecY protein and indicate where pBpA was incorporated. If known, mutants and their phenotypes are listed. For details on the phenotypes, see Refs (23,32,34). The indicated cross-linking products have been identified by western blotting. ‘+’ indicates a strong cross-linking product, ‘(+)’ a weaker cross-linking product and ‘−’ indicates that no interaction was observed.

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and the positions were selected on the basis of their conservation, their accessibility and based on mutant analyses (32–34). In particular, we choose residues for which amino acid replacements had been shown to have only a minor effect on protein transport at 37◦ C, but caused a more severe transport defect at 25◦ C or 42◦ C. These amber-stop codon-containing SecY constructs were subsequently expressed from a plasmid in E. coli cells carrying a pBpA-specific suppressor tRNA (31). Cells were grown in minimal media in the presence of pBpA and exposed to ultraviolet (UV) rays for activating pBpA. After UV exposure, cells expressing SecY with pBpA at the indicated position were fractionated and SecY was purified by metal affinity chromatography. As controls, the same procedure was repeated using cells, which had not been UV treated, and with cells expressing wild-type SecY without an amber-stop codon. UV-dependent cross-links were then identified by immune detection and by mass spectrometry. The C5 loop of SecY appears to be an important docking site for cytosolic partner proteins and, in particular, residues 357 and 363 have been shown to be important for SecY function (Table 1). We therefore tested SecY derivatives carrying pBpA at positions 356, 357 and 363 within the C5 loop for a possible FtsY contact (Figure 2A,B). These SecY derivatives were purified and possible cross-links to FtsY were analysed by immune detection. Western blotting using α-FtsY antibodies revealed that FtsY efficiently co-purified with SecY in all samples (Figure 2A), which further supports our previous conclusion of a direct SecY–FtsY interaction (23,24). In addition, the α-FtsY antibody recognized a strictly UV-dependent cross-linking product of about 130 kDa (Figure 2A), which was observed for SecY357pBpA and SecY363pBpA but not for SecY356pBpA or wild-type SecY. For SecY357pBpA, additional weaker bands at about 200 and 160 kDa were also recognized by the antibodies (Figure 2A). A weak band at about 170 kDa was recognized independently of the UV exposure and most likely represents a protein that co-purifies with SecY. The most prominent cross-linking product had a molecular mass of about 130 kDa, which is in agreement with the estimated mass of an SecY–FtsY cross-link. For identifying additional cross-linking products and for determining whether the weaker bands at 200 and 160 kDa also represented FtsY–SecY cross-links, SecY357pBpA was purified from UV-treated or nontreated cells and probed with antibodies against SecY. This revealed multiple UV-dependent cross-linking bands (Figure 2C), which were further analysed by mass spectrometry. SecY357pBpA was separated on SDS–PAGE and after Coomassie staining, the gel was cut into slices. These slices were subsequently subjected to ingel trypsin digestion and analysed by high-performance liquid chromatography (HPLC)–electrospray mass spectrometry. FtsY peptides were identified at 193, 167 and 123 kDa (Figure 2D, Table 2), which is in agreement with 566

the immune detection (Figure 2A). The integrated intensity was highest for the 193-kDa band, while the α-FtsY antibodies primarily recognized the 123-kDa band. Whether this reflects differences in the accessibility of the FtsY epitope is currently unknown. Different electrophoretic mobilities have also been observed for SecA–SecY crosslinks (35) and in agreement with this, we find SecA peptides both at 170 and 130 kDa (Figure 2C,D). The upper band most likely represents a cross-link of SecY to the N-terminus of SecA, whereas the lower band depicts a cross-link to the C-terminal part of SecA (35). Whether the presence of multiple FtsY–SecY cross-linking bands also reflects different binding modes is currently unknown. However, it is important to emphasize that FtsY displays a highly irregular migration behaviour on SDS–PAGE; the predicted molecular mass of FtsY is 56 kDa but it runs at about 100 kDa on SDS–PAGE (13,17) and even small modifications within the highly charged N-terminal A domain of FtsY can cause unpredictable changes in its running behaviour (18). In addition, FtsY also exists as an N-terminally truncated isoform of about 75 kDa (FtsY-14), which lacks the first 14 amino acids (13,17). Mass spectrometry (MS) also identified many ribosomal subunits in the SecY357pBpA sample (Figure 2C). Based on the size of detected bands, they most likely reflect cross-links between SecY and these ribosomal subunits, although we cannot entirely exclude that ribosomes also co-purify with SecY under the conditions tested. Nevertheless, the detection of ribosomal subunits further highlights the importance of the C5 loop of SecY for cotranslational targeting. In addition, SecY357pBpA efficiently cross-linked to the ribosome-associated chaperone trigger factor (TF), to the ATP-dependent RNA helicase SrmB and to the uncharacterized protein YniA. The weak band at 170 kDa, which was detected in both UV-treated and non-treated cells (Figure 2A), corresponds to the 170-kDa protein RNaseE (Figure 2C), which obviously co-purified with SecY. Because FtsY was efficiently cross-linked to residues SecY357 and SecY363, we extended the cross-linking approach to positions 238 and 240, located in the cytoplasmic loop C4, position 351 within the C5 loop and one position within the C-terminal C6 loop (SecY429) of SecY (Figure 3A,B). All these residues have been shown to be important for SecY function in previous mutagenesis studies (Table 1). Although FtsY co-purified with these SecY samples, we did not observe an SecY–FtsY cross-link for these positions (Figure 3A). We noticed one additional band, which was recognized by FtsY antibodies in the purified SecY429pBpA sample (Figure 3A, question mark), but this band only appeared without UV exposure and was therefore not further analysed. RNaseE was also weakly detectable in these samples. Residues 238 and 240 within the C4 loop are not very exposed but rather reside close to the membrane surface (Figure 3B) (36). Therefore, it appeared likely that these residues are involved in the interaction with the membrane-bound SecE and SecG

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FtsY–SecY Translocon Interaction A

B

C

D

Figure 2: FtsY binds to the fifth cytosolic loop of SecY. E. coli cells expressing either wild-type (wt) SecY or SecY with pBpA incorporated at the indicated positions within the C5 loop were harvested; one aliquot was UV exposed (+UV) while a second aliquot was kept in the dark (-UV). After cell fractionation, SecY was solubilized with 1% Triton-X-100 and purified via metal affinity chromatography. A) His-tag-purified SecY containing pBpA at different positions within the fifth cytosolic loop (100 μg protein) was separated on a 5–15% SDS–PAGE and after western transfer, probed with α-FtsY antibodies. Endogenous FtsY and an N-terminal truncated FtsY derivative (FtsY-14), which co-purified with SecY, are indicated. The SecY–FtsY cross-linking products are marked (*). The weak band at 170 kDa marked with ± corresponds to RNaseE, which co-purified with SecY (see also Figure 2C), and which cross-reacts with the polyclonal FtsY antibody. B) The cartoon shows the structure of the Thermus thermophilus Sec translocon (PDB 2ZQP). SecY is shown in green and SecE in grey. The cartoon was created using PyMol and the residues where pBpA was incorporated are marked in magenta. C) Cells expressing SecY357pBpA were isolated and either UV treated or kept in the dark. After cell fractionation, a crude membrane pellet was solubilized with 0.1% DDM and separated on SDS–PAGE. After western blot, the sample was decorated with α-SecY antibodies. The cross-linked bands were subsequently identified by mass spectrometry. Note that for the mass spectrometric analysis of SecY samples, we had to use 0.1% DDM as a detergent instead of Triton-X-100 and therefore FtsY did not co-purify with SecY in this experiment. # indicates that RNaseE was not cross-linked but efficiently co-purified with SecY. D) Mass spectrometric identification of in vivo cross-linking products of E. coli cells expressing SecY357pBpA. The material shown in (C) was separated on SDS–PAGE and Coomassie stained. Upper graph: optical density profile of the gel lane after background correction (right axis, bold line) and molecular weight (MW) interpolation (left axis, thin line and open circles) from positions of MW markers (filled diamonds). Lower graphs: mass spectrometric identification of SecA and FtsY following in-gel digestion by trypsin. Normalized distribution profiles (for normalization levels, see Table 2) from HPLC-MS-extracted ion chromatograms for peptides of SecA (continuous line) and FtsY (dashed line).

subunits of the SecYEG translocon (37). In agreement with this, we found for position SecY240pBpA a cross-link to SecE (Table 1). For testing more exposed residues within the C4 loop, pBpA was incorporated at positions 250, 255 and 259 of SecY and possible FtsY cross-links were identified by

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western blotting (Figure 3C,D). UV-dependent cross-links to FtsY were observed for positions 255 and 259, but not for position 250 (Figure 3C), which shows that FtsY is also in contact with the C4 loop of SecY. However, the crosslinking efficiency appeared to be significantly lower than with residues 357 and 363 of the C5 loop and was only detected after long exposure of the western blot. Positions 567

Kuhn et al. Table 2: Mass spectrometric identification of SecY–SecA and SecY–FtsY in vivo cross-linking products Protein name SecA FtsY

Protein IDa

Gel MWb (kDa)

MWc (kDa)

Number of amino acids

Coveraged (%)

Maximum intensitye

Number of peptidesf

P10408 P10121

173/130 193/167/123

102.0 56

901 497

71.0 38.4

1.8E+07 5.3E+04

124 14

The Coomassie-stained gel of purified SecY357pBpA shown in Figure 2D was cut into slices followed by in-gel trypsin digestion. Mass spectrometry was performed as described in Materials and Methods. a Identifier from Uniprot database. b Molecular weight for gel slice as determined by extrapolation. c Calculated molecular weight of mature protein. d Sequence coverage of total sequence by peptides detected. e Maximum intensity (sum of peptide integrated ion chromatograms). f Number of non-redundant peptides detected.

A

B

C

D

Figure 3: FtsY binds also to the fourth cytosolic loop of SecY. E. coli cells expressing SecY with pBpA incorporated at different position within the C4, C5 and C6 loops were treated as described in Figure 2. Endogenous FtsY and an N-terminal truncated FtsY derivative (FtsY-14), which co-purified with SecY, are indicated. A) SecY positions which had been identified in previous mutagenesis screens as being important for SecY function were analysed for FtsY interaction. The question mark indicates an uncharacterized band that is recognized by FtsY antibodies in the SecY429pBpA mutant and which disappears after UV exposure. RNaseE was not cross-linked to SecY but co-purified and was unspecifically detected by the polyclonal FtsY antibodies. B) Cartoon showing the T. thermophilus structure as in Figure 1; note that position 429 was not resolved in this structure. Residues where pBpA was incorporated are marked in magenta. C) SecY–FtsY cross-links were analysed for surface-exposed residues within the C4 and C5 loops of SecY. The SecY–FtsY cross-links are marked (*). Their detection required significantly longer exposure, suggesting that the cross-linking efficiency for these particular SecY residues was rather weak. Due to the long exposure, the co-purifying RNaseE band became very prominent. D) As in (B), position 255 was not resolved in this structure and therefore this residue is not indicated in the cartoon.

568

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255 and 259 in the C4 loop and positions 357 and 363 in the C5 loop are highly exposed and therefore predestined for making contact to FtsY. Equally surface exposed is also residue 347 in the C5 loop, but here we did not observe a cross-link to FtsY (Figure 3C). Owing to the longer exposure of the western blot, the co-purifying RNaseE band became very prominent for the SecY derivatives 250, 255, 259 and 347 (Figure 3C). The possible significance of this co-purification was not further analysed, but it is important to note that RNaseE obviously co-purified with all SecY derivatives and that this co-purification was observed in both dodecyl maltoside (DDM)-solubilized samples (cf. Figure 2C) and Triton-X-100-solubilized samples (cf. Figure 3A and C). We did not observe cross-links to FtsY when pBpA was incorporated into the N-terminal C1, C2 and C3 loops of SecY (Table 1). These loops are less exposed than the C4–C6 loops (cf. Figure 4E), but have been shown to bind to SecA, the motor protein of the post-translational protein transport (35,37,38). The incorporation of pBpA at SecY positions, which had been shown to be important for SecY function, could in principle promote or prevent relevant interactions of SecY. Although mutating the residues selected for pBpA incorporation did not cause a significant phenotype at the temperature used for growing the E. coli cells for in vivo cross-linking (30◦ C), we further verified the functionality of SecYpBpA constructs. The cold-sensitive E. coli SecY mutant strain SecY39 (32) is drastically impaired in growth at 25◦ C but grows almost normally at 37◦ C (Figure S3). To test the functionality of the SecYpBpA constructs, we transformed these plasmids into the SecY39 strain and tested growth at 25◦ C and 37◦ C in the presence or absence of pBpA (Figure S3). At 25◦ C in the absence of pBpA, significant growth was only observed for SecY39 carrying the wild-type SecY. In the presence of pBpA, however, all pBpA-containing SecY derivatives were able to support the growth of SecY39, albeit at different levels. In particular, SecY347, SecY363 and SecY429 displayed reduced complementation activity, which probably reflects the importance of these positions for SecY function. Nevertheless, despite these differences, all SecY amber-stop codon mutants were able to support the growth of SecY39 at 25◦ C in the presence of pBpA, indicating that they are functional. In summary, these in vivo cross-linking data clearly show that FtsY specifically interacts with the cytosolically exposed C4 and C5 loops of SecY. The binding site comprises residues which had been shown to be important for SecY function (SecY357 and SecY363), but also residues which so far have not been identified in mutagenesis screens as being important for SecY function (SecY255 and SecY259).

FtsY, SecA and the ribosome have overlapping binding sites on the SecY translocon Owing to their accessibility, the C4 and C5 loops appear to be major contact sites for SecY-interacting Traffic 2011; 12: 563–578

proteins (21,35,36,39) and we therefore analysed whether the pBpA-containing SecY derivatives cross-linked also to other proteins. The MS analyses had already revealed that SecY357 is not only in close contact to FtsY but also to SecA and the ribosome (Figure 2). This was confirmed by western blotting, which revealed a strong cross-link to SecA at 170 kDa and a weaker cross-link at 130 kDa (Figure 4A), in agreement with the positions at which SecA was identified by MS (Figure 2C,D). By western blotting, we also detected cross-links to the ribosomal protein L23 (Figure 4A), which is located at the ribosomal exit tunnel and which was also identified by MS (Figure 2C). We observed no cross-links to SecA or L23 for the neighbouring residues SecY356 and SecY363 (Figure 4A). Of the residues SecY238, SecY240, SecY351 and SecY429, which are important for SecY function (Table 1), we observed only for residue 429 a clearly detectable SecA–SecY cross-link and a very weak signal for residue 351 (Figure 4B). For position SecY429, we only observed the SecA cross-link at 130 kDa, which is in agreement with the data of Mori and Ito (35). A strong SecA–SecY cross-link was detected for the surface-exposed residue 347 and weaker cross-links for positions 250, 255 and 259 (Figure 4C). For the latter three positions, both the 130- and the 170-kDa crosslinking products were detectable. Both cross-link bands were comparable in strength for positions 250 and 255 of SecY, but for position 259, primarily the lower band was detected. The high-mobility SecA–SecY band probably reflects an SecY cross-link to the C-terminus of SecA, while the low-mobility band most likely represents a crosslink to the N-terminus of SecA (35). SecA binds via its polypeptide cross-linking domain (PPXD) to the C4 loop and this domain undergoes large conformational changes upon SecY binding (39), which could also contribute to the differences in the migration behaviour. We also tested the N-terminal loops of SecY for SecA interaction and found in agreement with previously published data (35,37) a strong SecA–SecY cross-link for position 111 in the C2 loop (Figure 4D). For the same residue, we also observed a cross-link to SecG (Table 1), while no SecA cross-links were observed for residue 11. In addition, we found an SecG cross-link for position 179 in the C3 loop. Surprisingly, by western blotting we observed cross-links to the ribosomal subunit L23 only for position 357 of SecY (Figure 4A, Table 1), although the C4 and C5 loops have been identified in both genetic (40) as well as in structural studies as important ribosome contact sites (41,42). We also used antibodies against L24 and L29, which are like L23 located at the ribosomal exit tunnel, but also found ribosome contact only for SecY357 (Figure 2C). However, it is mainly the 23S rRNA which contacts SecY (42,43) and possible rRNA–protein contacts were not analysed in our in vivo study. One additional reason for obtaining ribosome contact for only one SecY position is that in E. coli less than 10% of the ribosomes are membrane bound (44), while most of SecA (45) and FtsY (19) are localized at the membrane. 569

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D

E

B

C

Figure 4: The binding sites for FtsY, SecA and the ribosome on the SecY translocon partially overlap. A–D) The same material analysed for SecY–FtsY interaction in Figures 2 and 3 was also analysed with antibodies against the ATPase SecA and the ribosomal protein L23, which is located at the ribosomal exit tunnel. Because immune detection identified a cross-link to L23 only for residue 357, only this western blot is shown. With the exception of Figure 4D, only UV-treated samples are shown. Note that the western blot in (B) required significantly longer exposure for detecting even weak signals. For some residues, SecA antibodies recognized two cross-linking products. E) Cartoon showing the residues 11, 111 and 179 of the T. thermophilus SecY.

pBpA has a very short spacer length of 0.3–1.4 nm (46), which shows that FtsY is like SecA and the ribosome located in very close proximity to the cytosolic loops C4 and C5 of SecY. The exact SecY contact sites of SecA and FtsY as deduced from the in vivo cross-linking also explain the different phenotypes which have been observed for 570

some SecY mutants (Table 1). In the SecY39 (R357H) mutant, both the SecA-dependent translocation (32) as well as the SRP-dependent integration are impaired (23) and in agreement with this we found cross-links to both SecA and FtsY at this particular position. In contrast, the SecY40 (A363T) mutant does not impair SecA-dependent

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translocation but strongly inhibits SRP-dependent transport (23), which can be explained by our observation that only FtsY but not SecA is cross-linked to this position. The opposite phenotype is observed in the SecY205 (Y429D) mutant, which blocks the SecA pathway but not the SRP pathway (47,48). For this position, we found a cross-link to SecA but not to FtsY. We did not find any crosslinks for position 356, which appears to be rather tolerant towards mutations (34) and which is probably not crucial as a contact site for cytosolic partner proteins. We did also not detect FtsY, SecA or L23 cross-links to positions 11, 179, 238, 240 or 351 of SecY. Position 240 is at the SecY–SecE interface and in the corresponding mutant (SecY24), the stability of the SecYEG translocon is reduced, which explains that both the SRP and SecA pathways are impaired. The C2 and C3 loops of SecY are probably involved in binding to the non-essential SecG subunit of the bacterial Sec translocon (37) and in agreement with this, we found cross-links to SecG for positions 111 and 179 (Table 1). SecA has also been shown to bind to the N-terminus of SecY in binding assays (38) and cross-linking assays (35,37), and we found a strong SecA cross-link for position 111 of SecY. Finally, although residues 238 (C4 loop) and 351 (C5 loop) have been shown to be important for membrane protein biogenesis, they are suggested to be involved in a post-targeting step (34), which could explain the lack of FtsY cross-links for these positions. In summary, SecA is in contact with C2, C4, C5 and C6 of SecY (Table 1) and thus, its binding sites partially overlap with the binding site of FtsY, which binds also to the C4 and C5 loops. The SecA- and SRP/FtsY-dependent protein targeting pathways in E. coli operate predominantly in a non-overlapping mode (49,50), although SecA is required for complete integration of SRP-dependent membrane proteins with large periplasmic loops (51). A simultaneous binding of FtsY and SecA to SecY is difficult to envision, but instead FtsY and SecA probably compete for SecY access and it will be important to determine how bacterial cells co-ordinate FtsY/SecA access to the SecYEG translocon. However, the existence of multiple SecA-binding sites of different affinity in SecY (35,37,52) could in principle allow for a non-exclusive binding of FtsY and SecA to SecY.

the A domain in E. coli FtsY still allows for cotranslational targeting both in vivo (53) and in vitro (17), albeit with reduced efficiency. In contrast, the N and G domains are highly conserved and absolutely essential for FtsY function because they provide the contact site for SRP and regulate the GTP-dependent interactions of FtsY (54,55). The NG domain of FtsY is also highly homologous to the respective NG domains of SRα and Ffh/SRP54, but so far only FtsY has been shown to interact directly with the SecY translocon. Thus, it appeared possible that the FtsY–SecY interaction is mediated via the A domain. For testing this, we incorporated pBpA at multiple positions within the A domain between both amphipathic helices (Table 3), and analysed possible cross-links by western blotting. Antibodies against the C-terminal His-tag of FtsY recognized multiple UV-dependent bands for FtsY143pBpA, FtsY150pBpA, FtsY153pBpA and FtsY176pBpA (Figure 5A). In particular, we noticed a strong band at approximately 200 kDa, which was detected in all four FtsY derivatives. This band was also recognized by polyclonal α-SecY antibodies (Figure 5B). For FtsYpBpA143, SecY antibodies also recognized UV-dependent bands at 160 and 250 kDa (Figure 5B). A UV-independent band of 170 kDa was detected in all FtsY derivatives. Importantly, the 200- and 160-kDa FtsY–SecY cross-links were observed also when pBpA was incorporated into SecY (Figure 2), which provides an Table 3: Mapping the FtsY interactions by in vivo cross-linking Cross-links to FtsY domain A domain

A–N interface

N domain

The N-terminal A domain of FtsY is involved in SecY binding FtsY consists of three domains; the N-terminal A domain displays only low sequence conservation and has been shown to be involved in membrane binding of FtsY (17,18), but the exact role of this 197-amino acid long domain is still enigmatic. It is suggested that the A domain is highly flexible, which prevented so far successful crystallization attempts of full-size FtsY. Only an amphipathic lipid-binding helix has been identified at the N-terminus of the A domain (amino acids 1-14) (17,18), which is connected via a presumably unstructured 182-amino acid long stretch of unknown function to a second amphipathic lipid-binding helix at the interface between the A and N domains (amino acids 196-206) (16–18). The A domain is not present in all bacterial FtsY homologues and deleting Traffic 2011; 12: 563–578

G domain

Residue

SRP

TF

SecY

67 107 143 150 153 176 196 197 201 225 229 230 233 338 346 355 374 436 486

− − − − − − − − − +a +a,b − +a +a +a +a +a − −

− − n.t. − n.t. n.t. − − − +a +a,b − (+)a n.t. n.t. n.t. n.t. − −

− − +a +a,b +a +a − − − − − − − − − − − − −

Positions correspond to the amino acid numbering of the E. coli FtsY protein and indicate where pBpA was incorporated. The corresponding domains of FtsY are also indicated. ‘+’ indicates a strong cross-linking product, ‘(+)’ a weaker cross-linking product and ‘−’ indicates that no interaction was observed. a Cross-linking products were identified via immune detection. b Cross-linking products were identified via mass spectrometry. n.t., not tested.

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Figure 5: The A domain of FtsY interacts with the SecY translocon in vivo. pBpA was incorporated at different positions within the A domain of FtsY and samples were analysed as described in Figure 2. A) Cross-linking partners of the A domain were identified by antibodies against the C-terminal His-tag of FtsY. B) As in (A), but the western blot membrane was decorated with polyclonal α-SecY antibodies. The FtsY–SecY cross-links are marked with (*). C) Coomassie stained SDS–PAGE of cells expressing FtsY carrying pBpA at position 150 and exposed to UV. Note that in comparison with the western blot analyses, a gel of larger dimensions was used for the MS analysis. Upper graph: optical density profile of the gel lane after background correction (lower axis, bold line) and molecular weight (MW) interpolation (upper axis, thin line and open circles) from positions of MW markers (filled diamonds). Lower graphs: mass spectrometric identification of proteins following in-gel digestion by trypsin. Normalized distribution profiles (for normalization levels, see Table 4) from HPLC-MS-extracted ion chromatograms.

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additional proof for the significance of these cross-links. Additional cross-linking partners of FtsY were identified by mass spectrometry. FtsY150pBpA was purified from UV-treated cells and co-purifying or cross-linked bands were visualized by Coomassie blue staining. FtsY without amber-stop codon was also purified from UV-treated cells and used as a control to differentiate between co-purifying and cross-linked proteins. Identifying integral membrane proteins by MS has been notoriously difficult; nevertheless, we identified SecY peptides in the UV-dependent 200-kDa band (Figure 5C) and also in the 160-kDa band observed for FtsY-pBpA143 (Table 4), with a sequence coverage of about 7.5% of the total SecY amino acid sequence. Surprisingly, neither by western blotting nor by MS we were able to detect the prominent 130-kDa FtsY–SecY cross-link band that was detectable when pBpA was incorporated in SecY (Figures 2 and 3). This could indicate that the 130-kDa band reflects a different mode of SecY–FtsY interaction, e.g. to the conserved NG domain. Although this needs to be further analysed, a precedent for such differences in SecY–ligand interaction has been shown for the SecY–SecA interaction (35). The 250-kDa band was identified as a cross-link to elongation factor EF-Tu (Figure 5C). Because EF-Tu is the most abundant protein in E. coli, this interaction is probably unspecific. The UV-independent band at 170 kDa was identified as RNaseE, which also co-purified with SecY (Figures 2 and 3). Whether the co-purification of RNaseE with both FtsY and SecY is physiologically relevant needs to be determined in future studies. In summary, our combined western blotting and MS data clearly show that the A domain of FtsY interacts with the SecY translocon. We also tested several FtsY residues at the A–N interface and within the N and G domains. We choose residues within the NG domain of FtsY, which were not in immediate contact with Ffh, as deduced from the Thermus aquaticus FtsY(NG)–Ffh(NG) structure (56). For all residues tested, we did not detect cross-links to SecY by western blotting (Table 3). Instead, by using αHis antibodies, we observed a strong and almost exclusive cross-linking product of about 250 kDa for FtsY229pBpA and other residues within the N and G domains (Figure 6). The molecular mass of this cross-linked band was about identical to the FtsY–EF-Tu cross-link observed for residues within the A domain (Figure 5). However, for the A domain we observed multiple interactions (cf Figure 5A), while the 250-kDa band was the only prominent cross-link within the NG domain (Figure 6A) and it was therefore further analysed. Because the N and G domains of FtsY are required for SRP binding, we tested antibodies against Ffh, the protein component of the bacterial SRP. This antibody clearly detected the 250-kDa band (Figure 6A), which indicated that this band represents an FtsY–Ffh cross-linking product. As observed above, the molecular mass of the cross-linking product was significantly higher than expected for an FtsY–Ffh cross-link. Therefore, we repeated the in vivo cross-linking approach with two FtsY

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FtsY–SecY Translocon Interaction Table 4: Mass spectrometric identification of FtsY–SecY in vivo cross-linking products Position of pBpA FtsY-150 FtsY-150 FtsY-143

Identified protein

Protein IDa

Gel MWb (kDa)

MWc (kDa)

Number of amino acids

Coveraged (%)

Maximum intensitye

Number of peptidesf

FtsY SecY SecY

P10121 P0AGA2 P0AGA2

120 199 157

56 48.5 48.5

497 443 443

92.0 7.5 7.5

1.2E+08 8.6E+03 1.2E+04

235 2 2

The Coomassie-stained gel of purified FtsY150pBpA shown in Figure 5C and FtsY143pBpA (not shown) was cut into slices followed by in-gel trypsin digestion. Mass spectrometry was performed as described in Materials and Methods. a Identifier from Uniprot database. b Molecular weight for gel slice as determined by extrapolation. c Calculated molecular weight of mature protein. d Sequence coverage of total sequence by peptides detected. e Maximum intensity (sum of peptide integrated ion chromatograms). f Number of non-redundant peptides detected.

mutants, which had been shown to be impaired in their SRP interaction. The FtsYK399A mutant binds SRP only at unphysiological high SRP concentrations (7,57), while the FtsYA335W mutant binds SRP but their reciprocal GTPase activation is inhibited and therefore a stable FtsY–SRP complex is formed (7). When pBpA was incorporated in position 229 in the FtsYA335W and FtsYK399A mutants, the 250-kDa cross-link band was detectable in FtsYA335W but not in the FtsYK399A mutant (Figure 6A), which supports the conclusion that the 250-kDa complex represents an FtsY–Ffh cross-link. In the FtsYA335W mutant, the band detected by Ffh antibodies was only slightly enhanced (Figure 6A), which is expected because of the limited concentration of the endogenous Ffh. We analysed the presence of SRP in the 250-kDa in vivo cross-linking band also by MS and clearly identified Ffh in the 250-kDa band (Figure 6B, Table 5). This approach also detected the ribosome-associated chaperone Trigger Factor (TF) in the 250-kDa band (Figure 6B, Table 5) and EF-Tu at 230 kDa (data not shown). The presence of TF in the 250-kDa band was also verified by western blotting (Figure 6A). It is important to emphasize that FtsY229pBpA contains only a single photoreactive residue and therefore the 250-kDa complex does not reflect a ternary FtsY–Ffh-TF complex. Because TF and SRP bind non-exclusively to the ribosome via L23 (58), the proximity between FtsY and TF is probably because of binding of FtsY to the ribosome-bound SRP. In agreement with this notion, TF was detectable in the 250-kDa band in wild-type FtsY and in the FtsYA335W mutant but not in the FtsYK399A mutant, which is unable to interact with SRP (Figure 6A). In a previous study, it has been suggested that upon FtsY–SRP interaction at the ribosome, TF is dispelled (59), while our data now indicate that TF does not necessarily dissociate from the ribosome after SRP–FtsY contact. This discrepancy is most likely explained by the fact that in our experiments FtsY is in its native, membrane-bound state (19), while the other study was performed with soluble FtsY in the absence of membranes. In addition, the cross-linking approach can only monitor a small fraction of the interacting proteins, while in the other study the bulk of material was analysed.

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In summary, the 250-kDa band represents a cross-link between FtsY and either Ffh or TF, which are both located at the ribosomal subunit L23 and which both have a molecular mass of approximately 49 kDa. FtsY–Ffh cross-links were observed for multiple residues within the N and G domains of FtsY (Table 3), which are in the crystal structure not recognizable as immediate contact sites to Ffh. In particular, the distances between FtsY residues 225-233 and SRP are close or beyond the pBpA spacer length (Figure 6C). In the crystal structure, FtsY374 is also too far away from Ffh to be crosslinked by pBpA, but we still observe an Ffh cross-link to this position. It is important to note that the Xray structure was determined for the respective NG domains, but without the substrate-binding domain of Ffh (the M domain) and without the A domain of FtsY. Thus, it is possible that the FtsY residues contact the M domain of Ffh, which was also suggested in a previous analysis (60) In addition, the FtsY–Ffh complex has been shown to be highly dynamic (7) and the X-ray structure depicts only one of probably multiple conformations. Our non-synchronized in vivo cross-linking approach probably detects these multiple conformations, which can further explain these unexpected cross-linking results. The same residues within the N domain of FtsY, which gave crosslinks to Ffh, were also cross-linked to TF (Table 5), which supports the assumption that FtsY is close to TF only after binding to ribosome-bound SRP. Ffh cross-links were not obtained for residues within the A domain of FtsY (Table 3), which is in agreement with the fact that the A domain is not universally conserved and not absolutely essential for SRP-dependent protein targeting. Our in vivo cross-linking study shows that FtsY binds to the membrane-exposed C-terminal loops of SecY, which serve as a docking site for multiple ligands like the ribosome or SecA (36,39,42). By binding to the cytosolic loops 4 and 5 of SecY, FtsY would be perfectly positioned to align the incoming SRP–RNC with the protein conducting channel. This is very similar to the SecA–SecY binding, which also aligns the proposed substrate binding clamp of SecA with the SecY channel (39). However, 573

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Figure 6: The NG domain of FtsY cross-links to SRP and TF. A) Wild-type FtsY or the FtsY mutants FtsYA335W and FtsYK399A, carrying pBpA at position 229, were expressed and processed as described in Figure 2. Samples were analysed by western blotting using antibodies against the C-terminal Histag of FtsY (upper panel), against Ffh, the protein component of the bacterial SRP (middle panel), and against the ribosomeassociated chaperone TF (lower panel). B) Mass spectrometric determination of proteins in a lane from SDS–PAGE of UV-treated cells expressing FtsY229pBpA after staining with Coomassie Brilliant Blue. Upper graph: optical density profile of the gel lane after background correction (right axis, bold line) and molecular weight (MW) interpolation (left axis, thin line and open circles) from positions of MW markers (filled diamonds). Lower graphs: mass spectrometric identification of proteins following in-gel digestion by trypsin. Normalized distribution profiles (for normalization levels, see Table 5) from HPLC-MS-extracted ion chromatograms for peptides of TF (dotted line), Ffh (continuous line) and FtsY (dashed line). C) Cartoon showing the FtsY–Ffh complex of T. aquaticus (PDB 1OKK.). Positions in FtsY where pBpA was incorporated are shown in magenta and are labelled. Ffh, the protein component of SRP, is shown in yellow and FtsY in green.

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while SecA and its substrates can simultaneously interact with the SecY translocon, FtsY probably has to dissociate from the cytosolic loops to expose the RNC-binding site of SecY (4,41,42). This is strikingly analogous to the position of SRP on the translating ribosome. Here, SRP occupies the translocon-binding site of the ribosome (4,61,62) and only upon interacting with SR, the NG domain of Ffh is delocalized and the translocon-binding site of the ribosome is exposed (4). It is tempting to speculate that when FtsY interacts with SRP, this interaction not only causes a delocalization of SRP on the ribosome but also a delocalization of FtsY on the translocon, which would then perfectly position the ribosomal exit tunnel onto the protein conducting channel. However, this needs to be verified in future studies. The respective NG domains of SRα and FtsY exhibit high sequence conservation, but differ significantly within their N-terminal domains. The N-terminal SRX2 domain in SRα is required for the interaction with SRβ (63), but the exact function of A domain in FtsY has remained unclear. So far, only an N-terminal lipid-binding domain has been identified (17). Our observation that the A domain of E. coli FtsY is in contact with the SecY translocon now attributes an important function to the A domain of E. coli FtsY. However, deleting the A domain of E. coli FtsY does not completely inhibit FtsY function (16,53) and therefore it is possible that additional contact sites exist within the NG-domain of FtsY. This would explain why we observe a strong 130-kDa SecY–FtsY cross-link when pBpA is incorporated in SecY, but not with pBpA incorporated within the A domain of FtsY. Alternatively, we can also not exclude that the FtsY–SecY interaction is not essential for cotranslational targeting in bacteria. The A domain is not present in all prokaryotes; e.g. it is lacking in FtsY homologues of archaea and many gram-positive bacteria or it is at least significantly shorter than in many gramnegative bacteria (64–66). Because the SRP and SecA pathways in gram-negative bacteria compete for access to the SecY translocon, the presence of a SecY-binding site in FtsY could allow the aggregation-prone SRP substrates a preferential access to SecY. In contrast, this would not be required in archaea, which lack the SecA pathway, or in gram-positive bacteria, in which the SecA and SRP pathways seem to co-operate rather than compete during the delivery of substrates to the SecY translocon (67). Nevertheless, it will be important to determine whether FtsY in gram-positive bacteria or archaea uses a different mode of translocon interaction or whether in these species FtsY–translocon interaction is entirely dispensable. In summary, our data explain why cotranslational targeting in bacteria can function without the need of a SRβ subunit. The bacterial FtsY encompasses the functions of both the eukaryotic SRα and SRβ subunits in one single protein: it recruits the SRP–RNC to the membrane-like SRα and also co-ordinates their binding onto the Sec translocon, a function that is associated with the SRβ subunit in eukaryotes (62). However, it is important to

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FtsY–SecY Translocon Interaction Table 5: Mass spectrometric identification of FtsY–FFh and FtsY–TF in vivo cross-linking products Protein name FtsY Ffh TF

Protein IDa

Gel MWb (kDa)

MWc (kDa)

Number of amino acids

Coveraged (%)

Maximum intensitye

Number of peptidesf

P10121 P0AGD7 P0A850

110 256 256

56 49.7 48.2

497 435 432

92.0 82.6 28.7

2.6E+08 3.1E+07 4.0E+05

226 144 10

The Coomassie-stained gel of purified FtsY229pBpA shown in Figure 6C was cut into slices followed by in-gel trypsin digestion. Mass spectrometry was performed as described in Materials and Methods. a Identifier from Uniprot database. b Molecular weight for gel slice as determined by extrapolation. c Calculated molecular weight of mature protein. d Sequence coverage of total sequence by peptides detected. e Maximum intensity (sum of peptide integrated ion chromatograms). f Number of non-redundant peptides detected.

emphasize that SRβ interacts with the β-subunit of the eukaryotic translocon (21), which has no homologue in bacteria. Therefore, during evolution, the recruitment of SRP–RNCs and translocon binding were not just separated into two separate proteins; eukaryotes also adapted a different mode of recognizing an unoccupied translocon.

Materials and Methods Strains and cell growth The following E. coli strains were used: BL21, DH5α (68), SecY24 (30), SecY39 (32), IY28 (19) and SKP1101 (69) and they were grown in LB medium at 37◦ C unless noted otherwise. For plasmid and mutant construction, see Supporting Information.

In vivo cross-linking BL21 carrying the plasmids pSup-BpaRS-6TRN and pTrc99a-SecYHis EG/ pTrc99a-FtsYHis was grown according to the procedure described previously at 30◦ C in the presence of 1 mM pBpA (18). After UV exposure of whole cells, cells were broken in a French pressure cell and proteins were purified via Talon-Affinity-Resin (Clonetech). FtsYpBpA samples for MS were solubilized with 1% Triton-X-100 and SecYpBpA samples with 0.1% DDM. Purified samples were separated on SDS–PAGE and cross-linking products were identified after western blotting by immune detection.

Protein identification by HPLC–electrospray mass spectrometry Lanes of an SDS–PAGE were cut into horizontal 1-mm slices that were processed individually for establishing abundance profiles of identified peptides. Proteins were modified by iodoacetamide, in-gel digested and analysed by HPLC–electrospray MS using an LTQ-FT (Thermo Electron Corporation) or an LTQ-FT-Ultra (for SecY357) as described (70). The MS and MS/MS spectra were searched against E. coli protein sequences deposited at the UniProt database (release 15.5, 7 July 2009) using an in-house installation of the program OMSSA (version 2.1.4) (71) and quantified using MSQUANT (version 2.0) (72) as described (73). Peptide hits were considered significant if the precursor and product ion masses matched within 2 ppm and 0.5 relative mass units, respectively, and if the E value was below 0.01. Protein distribution profiles (74) were calculated as the sum of integrated extracted ion chromatograms for all peptides of one protein identified by MS/MS, which was then plotted against the position of the individual gel slice.

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Fluorescence microscopy and in vivo FRET analyses Cells expressing fluorescently labelled proteins were grown to exponential phase in M63 medium supplemented with amino acids (1 mM each) and thiamine (10 mg/L). After induction with 0.25 mM isopropylthio-galactopyranoside (IPTG) and/or 0.075% arabinose for 2 h, cells were immobilized on a microscope slide covered with 1% agarose in M63. FtsY A335W-expressing cells were grown with 1% glucose to repress expression prior to induction. Induction was subsequently performed with 1 mM IPTG and 0.2% arabinose to compensate for repression. Proteins were visualized using an Olympus BX-51 microscope at 100× magnification with a numerical aperture of 1.4 and a cyan fluorescent protein (CFP) or YFP filter set. Images were acquired with a charge-coupled device (CCD) camera (F-View, Olympus) and image processing was performed using IMAGEJ 1.42 (http://www.rsb.info.nih.gov/ij/). Acceptor photobleaching FRET measurements were performed as described before (25) on a custom-modi?ed Zeiss Axiovert 200 microscope. Cells were concentrated about 10-fold by centrifugation and applied to a thin agarose pad (1% agarose in tethering buffer [10 mM potassium phosphate, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM L-methionine, 10 mM sodium lactate, pH 7.0]). Excitation light from a 75 XBO lamp, attenuated by a neutral-density filter (depending on fluorescence intensity, neutral density 1.0, 1.6 or 2.0, respectively), passed through a band-pass (BP) 436/20 filter and a 495DCSP dichroic mirror and was reflected on the specimen by a Z440/532 dual-band beam splitter (transmission 465–500 and 550–640 nm; reflection 425–445 and 532 nm). Bleaching of YFP was accomplished by a short (20 seconds) illumination with a 532-nm diode laser (Rapp OptoElectronic), reflected by the 495DCSP dichroic mirror into the light path. Emission from the field of view, which was narrowed with a diaphragm to the area bleached by the laser, passed through a BP 485/40 filter onto a photo multiplier (Hamamatsu H7421-40; Hamamatsu). For each measurement point, photons were counted over 0.5 seconds using a counter function of the PCI-6034E board, controlled by a custom-written LABVIEW 7.1 program (National Instruments). The apparent value of FRET efficiency was calculated as a fractional increase in the cyan fluorescence upon bleaching of YFP, Cyan/Cyanpost-bleach , with Cyan being the change in signal in the CFP channel before and after bleaching and Cyanpost-bleach being the signal after bleaching. Values above 0.5% are considered to reflect a significant interaction (25). Importantly, thus the obtained values of the apparent in vivo FRET efficiency are substantially lower than the theoretical FRET efficiency for the same pair. This reduction is because of a contribution of the background resulting from cellular autofluorescence and from the excitation light scatter by cells to the overall signal in the cyan channel, which effectively reduces specific Background + Cyanpost-bleach . signal since Cyanpost-bleach = CyanCFP post-bleach

Expression and purification of proteins, GTPase assay pTrc99a-FtsY (18), pTrc99a-Ffh, pTrc99a-FtsY-Cer and pTRc99a-Ffh-YFP were transformed into E. coli DH5α cells (69). Cells were grown at 37◦ C

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Kuhn et al. and induced with 1 mM IPTG after reaching an optical density (OD)600 of 0.6–0.8 and subsequently harvested 4 h after induction. Proteins were ¨ affinity purified via their His-tags using an Akta chromatography system (GE Healthcare) and a 1-mL HisTrap FF crude nickel column (GE Healthcare) or by using Talon slurry (Clonetech). The equilibration/wash buffer contained 50 mM HEPES KOH pH 7.6, 1 M NH4 Ac, 10 mM MgAc2 , 1 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), 30 mM imidazol pH 7.8 and 10% glycerol. The elution buffer contained 50 mM HEPES KOH pH 7.6, 1 M NH4 Ac, 10 mM MgAc2 , 1 mM DTT, 0.5 mM PMSF, Roche Complete Inhibitor Cocktail Tablets, 400 mM imidazol pH 7.8 and 10% glycerol. Subsequently, the buffer was exchanged to 2× HT buffer (100 mM HEPES KOH pH 7.6, 200 mM KAc pH 7.5, 20 mM MgAc2 , 2 mM DTT) using PD10 columns (GE Healthcare). Ffh and Ffh-YFP were stored at −20◦ C in HT buffer supplemented with 50% glycerol, FtsY and FtsY-Cer at −70◦ C in HT buffer supplemented with 10% glycerol. GTPase assays were performed at 25◦ C in a total volume of 20 μL and contained 0.05 μM Ffh and FtsY. The reaction was performed in HT buffer (50 mM HEPES, pH 7.6; 100 mM KOAc, pH 7.5; 10 mM MgOAc and 1 mM DTT) and started by the addition of GTP [200 μM GTP + 2.5 μM [γ-P32 ]GTP (∼2.5 μCi)]. When indicated, 0.5 μg of 4.5S RNA was added. Aliquots were removed at frequent time intervals. The reaction was stopped on ice by the addition of 800-μL charcoal suspension (10% in 20 mM phosphoric acid) and the liberated phosphate in the supernatant after centrifugation was determined using a scintillation counter.

Acknowledgments This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG grants FOR 929 to V. S. and H.-G. K. and FOR 967 to H.-G. K.) and the Excellence Initiative of the German Federal and State Governments (EXC 294 bioss). E. S. was supported by the Hartmut-Hoffmann-Berling International Graduate School of Molecular and Cellular Biology. We thank David Braig for providing purified SRP and FtsY and Bettina Knapp for excellent technical assistance.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1: Characterization of the fluorescently labelled FtsY and Ffh derivatives. A) FtsY-Cer was expressed in the conditional FtsYdepletion strain E. coli IY28, in which the expression of the chromosomal ftsY copy depends on the addition of arabinose. Cells were grown on LB medium supplemented with either 0.4% arabinose or 0.4% fructose and growth was monitored in comparison with IY28 cells containing no plasmid, the empty vector pTrc99a (vector) or wild-type FtsY in pTRc99a (wt FtsY). All growth experiments were performed in the absence of any inductor, indicating that the basal expression level of the inducible plasmidborne copies is sufficient for complementation. B) Western blotting of cell extracts from FtsY-Cer-expressing cells, using α-FtsY antibodies. FtsY14/FtsY-14-Cer corresponds to N-terminally truncated FtsY derivatives, which lack the first 14 amino acids and show significantly enhanced electrophoretic mobility (15,16). C) Functionality of Ffh-YFP was tested in the temperature-sensitive Ffh mutant strain E. coli SKP1101. Growth was monitored on LB medium at 37◦ C or at the non-permissive temperature (42◦ C). D) Western blot of cell extracts from Ffh-YFP-expressing cells using α-Ffh antibodies. E) GTPase activity of wild-type Ffh and FtsY or the fluorescently tagged proteins was determined as described in Materials and Methods. Purified proteins were used at a final concentration of 0.05 μM and 4.5S RNA was added to a final concentration of 0.025 μg/μL when indicated. At least three independent experiments were performed. F) Possible cleavage of FtsY-Cer or Ffh-YFP was controlled by western blotting using α-GFP antibodies, which recognize both Cerulean and YFP. Extracts from cells expressing either FtsY-Cer, Ffh-YFP or Cerulean were analysed. Free Cer/YFP was undetectable in FtsY-Cer/Ffh-YFP-expressing strains, indicating that the fluorescent tag was not cleaved off. However,

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we did notice some proteolysis of Ffh-YFP, which did not result in free YFP. Figure S2: Activity of the fluorescently labelled SecY derivatives. A) The functionality of YFP-SecY and SecY-YFP was analysed in the temperature-sensitive SecY mutant strain E. coli SecY24. Cells were grown for 90 min at 30◦ C and then either shifted to the non-permissive temperature (42◦ C) or further incubated at 30◦ C. B) Western blot of DH5α cells expressing SecY-YFP or YFP-SecY. The size of SecY-YFP is slightly larger than the YFP-SecY version, because of a longer linker region. SecY indicates the endogenous SecY. C) Possible cleavage of YFP-SecY and SecY-YFP was analysed as in Figure S1. We observed no free YFP in YFP-SecY- or SecY-YFP-expressing cells. The cleavage product at 34 kDa in the SecY-YFP lane is still detected by the GFP antibody and therefore does not represent the free SecY. Figure S3: pBpA-containing SecY derivatives are functional. The functionality of the pBpA-containing SecY constructs was analysed by expressing them in the cold-sensitive SecY mutant E. coli SecY39. TAG stop codons were incorporated into the indicated SecY residues (SecY11SecY429) of pTRc99aSecY(His) EG and the plasmid-borne copies were transformed into SecY39. Growth was monitored at 37◦ C or at 25◦ C in the absence or presence of pBpA. At 37◦ C, plates were incubated for 12 h. For growth experiments at 25◦ C, all plates were preincubated at 37◦ C for 90 min and then transferred to 25◦ C and incubated for 36 h. pBpA was added to the LB plates at a concentration of 1 mM. Wt type SecY corresponds to the plasmid-borne copy of wt SecY and vector to the empty pTRc99a plasmid. All growth experiments were performed in the absence of inductor, indicating that the basal expression level of the inducible plasmid-borne copies is sufficient for complementation. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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