Heat Shock Transcription Factor σ32 Co-opts the Signal Recognition Particle to Regulate Protein Homeostasis in E. coli

The bacterial heat shock transcription factor, Ïƒ32, maintains proper protein homeostasis only after it is targeted to the inner membrane by the signal recognition particle (SRP), thereby enabling integration of protein folding information from both the cytoplasm and cell membrane.


Introduction
The heat shock response (HSR) maintains protein homeostasis (proteostasis) in all organisms. The HSR responds to protein unfolding, aggregation, and damage by the rapid and transient production of heat shock proteins (HSPs) and by triggering other cellular protective pathways that help mitigate the stress. Although the specific HSR is tailored to each organism, chaperones that mediate protein folding and proteases that degrade misfolded proteins are almost always included in the core repertoire of induced protein and are among the most conserved proteins in the cell. These HSPs maintain optimal states of protein folding and turnover during normal growth, while decreasing cellular damage from stress-induced protein misfolding and aggregation. Malfunction of the HSR pathway reduces lifespan and is implicated in the onset of neurodegenerative diseases in higher organisms [1][2][3].
In E. coli and other proteobacteria, s 32 mediates the HSR by directing RNA polymerase to promoters of HSR target genes [4][5][6][7][8][9]. Given the importance of this response and the necessity for a rapid but transient increase in expression of HSPs, it is not surprising that regulation of the HSR across organisms is complex. s 32 is positively regulated by a feed-forward mechanism in which exposure to heat melts an inhibitory mRNA structure enabling high translation of s 32 mRNA [10,11] and is negatively regulated by two feedback loops [12] mediated through members of the s 32 regulon ( Figure 1A). s 32 activity is coupled to the cellular protein folding state via a negative feedback loop executed by the two major chaperone systems, DnaK/J/GrpE and GroEL/S. There is extensive support for the model that free chaperones directly inactivate s 32 and that these chaperones are titrated by unfolded proteins that accumulate and bind chaperones during a HSR. Depletion of either chaperone system or overexpression of chaperone substrates leads to an increase in s 32 activity, and conversely, overexpression of either chaperone system decreases s 32 activity [13,14]. Inhibition is likely direct, as DnaK/J and GroEL/S bind s 32 in vitro and inhibit its activity in a purified in vitro transcription system [13,[15][16][17]. s 32 stability is controlled by the inner membrane (IM) protease FtsH: deletion of the protease stabilizes s 32 [18][19][20], and FtsH degrades s 32 in vitro, albeit slowly [18,20]. DnaK/J and GroEL/S also regulate stability, as their depletion leads to s 32 stabilization in vivo [13,14,21], although this finding has not yet been recapitulated in vitro [22].
Despite the regulatory complexity of the current model, it inadequately addresses two issues that are central to our understanding of the circuitry controlling the HSR, motivating us to search for additional players in the response: (1) Exhaustive genetic screens for mutations in s 32 that result in misregulation have identified a small cluster of four closely spaced amino acid residues (Leu47, Ala50, Lys51, and Ile54), of which three are surface exposed, as well as a somewhat distant fifth residue that abuts this patch in the folded s 32 structure. When these residues are mutated, cells have both increased level and activity of s 32 , indicating that this region is involved in a central process required for operation of the negative feedback loops that control both the activity and degradation of s 32 ( Figure 1A) [23][24][25]. However, the phenotypes of these mutants are not recapitulated in vitro, where both FtsH degradation and chaperone-mediated inactivation of mutant and WT s 32 are experimentally indistinguishable [25,26]. Thus, we do not understand how this ''homeostatic control region'' of s 32 functions. (2) s 32 is thought to monitor the folding status of IM proteins as well as cytoplasmic proteins, but the mechanism for this additional surveillance is unknown. Their close connection is indicated because (1) the IM protease, FtsH, not only degrades s 32 , but also maintains quality control in the IM by degrading unassembled IM proteins; (2) induction of the HSR is a very early response to perturbations in the co-translational membrane-trafficking system that brings ribosomes translating IM proteins to the membrane [27][28][29]; and (3) IM proteins are significantly overrepresented both in the s 32 regulon [30] and in an unbiased overexpression screen for HSR inducers [30].
In this report, we identify the co-translational protein targeting machinery, comprised of the Signal Recognition Particle (SRP; Ffh protein in complex with 4.5S RNA; Figure 2A) and the SRP Receptor (SR; FtsY), as a regulator of s 32 . We show that SRP preferentially binds to WTs 32 compared to a mutant s 32 with a defective homeostatic control region. We further show that a fraction of s 32 is associated with the cell membrane and that both the SRP-dependent machinery and the homeostatic control region of s 32 are important for this localization. Lastly, the regulatory defects in HSR circuitry caused by mutation of either the s 32 homeostatic control region or the co-translational targeting machinery are circumvented by artificially tethering s 32 to the IM. We propose that SRP-dependent membrane localization is a critical step in the control circuitry that governs the activity and stability of s 32 . Membrane localization is widely used to control s factors, but this is the first case where the IM-localized state is used for dynamic regulation rather than as a repository for an inactive protein.

A Transposon Insertion Mutant at the ftsY Promoter Region Is Defective in Feedback Control
To identify additional players involved in activity control of s 32 , we carried out a genetic screen for transposon mutants with increased s 32 activity under conditions that inactivate s 32 in wildtype cells (see Methods). To impose a condition that mimics the negative feedback control of s 32 , the DnaK/J chaperones were overexpressed from an inducible promoter at their chromosomal locus. Under these conditions, a s 32 -regulated lacZ chromosomal reporter (P htpG -lacZ) is expressed so poorly that cells do not make sufficient b-galactosidase to turn colonies blue on X-gal indicator plates. We screened for blue colonies, indicative of a defect in s 32 inactivation. A conceptually similar screen previously identified mutations in the DnaK/J chaperones-key negative regulators of the s 32 response [31]. In addition to re-identifying these components, we found an insertion in the promoter region of ftsY (pftsY::Tn5), located 39 bp upstream of the ftsY open reading frame. The pftsY::Tn5 strain had a 3-to 4-fold reduction in the level of FtsY, the SR, and a ,7-fold increase in the activity and amount of s 32 relative to WT (Table 1). Defects were complemented by a plasmid carrying ftsY. Unlike WT, in the pftsY::Tn5 strain s 32 activity did not respond to increased chaperone expression. Upon chaperone overexpression in WT cells, the specific activity (S.A.) of s 32 fell to 0.3, relative to that in cells growing without chaperone overexpression. In contrast, upon chaperone overexpression in pftsY::Tn5 cells, the S.A. of s 32 did not change, suggesting a defect in chaperone-mediated activity control in that strain (Table 1). This finding raised the possibility that the high activity of s 32 in pftsY::Tn5 resulted from disruption of activity control of s 32 , rather than reflecting a cellular response to accumulation of unassembled membrane proteins.

s 32 Directly Interacts with SRP
We tested whether s 32 binds to either FtsY (SR) or to Ffh, the protein component of SRP. Ffh is a two-domain protein, comprised of an M-domain that binds the signal sequence and 4.5S RNA, and an NG-domain that binds to SR, the ribosome,

Author Summary
All cells have to adjust to frequent changes in their environmental conditions. The heat shock response is a signaling pathway critical for survival of all organisms exposed to elevated temperatures. Under such conditions, the heat shock response maintains enzymes and other proteins in a properly folded state. The mechanisms for sensing temperature and the subsequent induction of the appropriate transcriptional response have been extensively studied. Prior to this work, however, the circuitry described in the best studied bacterium E. coli could not fully explain the range of cellular responses that are observed following heat shock. We report the discovery of this missing link. Surprisingly, we find that s 32 , a transcription factor that induces gene expression during heat shock, needs to be localized to the membrane, rather than being active as a soluble cytoplasmic protein as previously thought. We show that, equally surprisingly, s 32 is targeted to the membrane by the signal recognition particle (SRP) and its receptor (SR). SRP and SR constitute a conserved protein targeting machine that normally only operates on membrane and periplasmic proteins that contain identifiable signal sequences. Intriguingly, s 32 does not have any canonical signal sequence for export or membrane-integration. Our results indicate that membrane-associated s 32 , not soluble cytoplasmic s 32 , is the preferred target of regulatory control in response to heat shock. Our new model thus explains how protein folding status from both the cytoplasm and bacterial cell membrane can be integrated to control the heat shock response. and GTP (Figure 2A). We first used co-immunoprecipitation analysis. Interacting proteins were immunoprecipitated with antibodies against either FtsY or Ffh and, following resolution on SDS-PAGE, antibodies against s 32 or s 70 were used to probe for the presence of these proteins. s 32 was detected in the immunoprecipitations ( Figure 2B, lanes 7 and 8), and this signal was dependent on the presence of s 32 in the strain ( Figure 2B, lanes 1-4). By contrast, s 70 , although much more abundant than s 32 in the cell, did not interact with either SRP or SR ( Figure 2B, Lanes 3,4 and 7,8), indicating that interaction with SRP is not a general property of ss. It was not surprising that s 32 was co-immunoprecipitated with both SRP and SR, as the latter two components interact in vivo. To determine the direct binding partner of s 32 , purified Ffh and FtsY were resolved on SDS-PAGE, transferred to nitrocellulose, and incubated with purified s 32 . Antibodies against s 32 detected s 32 present at the molecular weight corresponding to Ffh but not SR ( Figure 2C). In a reciprocal experiment, purified s 32 was resolved on SDS-PAGE, transferred to nitrocellulose, and incubated with purified Ffh or SR. Ffh, but not SR, bound s 32 (unpublished data). Similar studies did not reveal an interaction between s 70 and either Ffh or SR (unpublished data). We determined which Ffh domain binds s 32 by partially-proteolyzing Ffh to produce an 18 kDa M-domain and a 38 kDa NG-domain, resolving the mixture by SDS-PAGE, transferring to nitrocellulose, and probing with s 32 . s 32 was detected at the position of full-length Ffh and the M-domain, but not at the position of the NG-domain ( Figure 2D), indicating that the M-domain contains the determinants mediating the s 32 -interaction.
We used in vivo crosslinking to validate the direct interaction of SRP (Ffh+4.5S RNA) and s 32 . We created a s 32 derivative with an N-terminal 66HIS-tag and a photoreactive amino acid analog (pBPA) at amino acid position 52 (66HIS-s 32 T52pBPA; see Methods), which is active as WTs 32 in expression of the s 32 reporter P htpG -lacZ (activity is 150% that of WT; within the range of the variability of the assay; unpublished data). Following UV irradiation of whole cells, anti-Ffh immunoblotting of the whole cell lysate detected one predominant crosslinked product, which was dependent on UV-irradiation ( Figure 3A, lanes 1 and 2) and pBPA at position 52 ( Figure 3A, lanes 2 and 4). This UV-and pBPA-dependent product was also detected with anti-s 32 immunoblotting ( Figure 3A, lane 6). To determine whether the crosslinked product represented 66HIS-s 32 T52pBPA-Ffh, we determined whether this product was identified both by coimmunoprecipitation with anti-Ffh antisera ( Figure 3B) and by affinity purification of 66HIS-s 32 T52pBPA on a TALON resin ( Figure 3C). Upon immunoprecipitation with anti-Ffh antisera, we detected a single higher molecular mass band, which reacted with both anti-Ffh ( Figure 3B, lane 2) and -s 32 ( Figure 3B, lane 6).  Upon affinity purification on a TALON resin, anti-Ffh identified the same predominant UV-and pBPA-dependent Ffh-containing crosslinked product (compare Figure 3B and 3C, lane 2). Importantly, no free Ffh was recovered following TALON purification, indicating that the recovery of the Ffh conjugate was mediated by the covalently linked 66HIS-s 32 , rather than interaction with either the TALON resin or another protein.
These results strongly suggest that s 32 directly interacts with Ffh in vivo. Although only a faint band was seen at the same position using anti-s 32 immunoblotting, this was likely a result of high background in this area of the gel, possibly because of extensive interaction between chaperones and s 32 ( Figure 3C, lanes 5-8).

I54Ns 32 Is Defective in Interacting with SRP
The function of the homeostatic control region of s 32 is not known [25]. I54Ns 32 is a mutation located in this region is severely compromised in both activity and degradation control, but the mechanism responsible for this phenotype had not yet been determined [25]. We therefore compared the binding of WTs 32 and I54Ns 32 to SRP using gel filtration. We incubated WTs 32 or I54Ns 32 either alone or in combination with SRP and subjected the mixture to gel filtration. Analysis of the elution profiles demonstrated that most WTs 32 was shifted towards the higher molecular weight region in the presence of SRP, and additionally, a fraction of s 32 eluted at a higher molecular weight than that of SRP alone, indicative of an SRP-s 32 complex [compare A 280 profiles of s 32 , SRP, and SRP-s 32 ( Figure 4A) with immunoblotting for s 32 ( Figure 4B; rows 1,2)]. s 32 present at a molecular weight between s 32 and SRP likely represents transient forms of the s 32 -SRP complex. In sharp contrast, an interaction between I54Ns 32 with SRP was almost undetectable [compare A 280 profiles of I54Ns 32 and SRP ( Figure 4A) with immunoblotting for I54Ns 32 ( Figure 4B; rows 3,4)], indicating that I54Ns 32 bound more weakly to SRP than WTs 32 . Neither WTs 32 nor I54Ns 32 interacted detectably with Ffh, indicating that differential binding is dependent on the formation of SRP (Ffh+4.5S RNA), the biologically relevant cellular species of Ffh.

s 32 Is Partially Membrane Associated in an SRP-Dependent Process
The biological function of SRP is co-translational protein targeting, leading us to test whether s 32 may be targeted to the IM through an SRP-dependent mechanism. Rapid degradation by FtsH normally keeps s 32 levels very close to the detection limit (,20-50 molecules/cell; [8]), making reproducible detection following fractionation very difficult. Therefore, we performed fractionation experiments ( Figure 5), either in cells expressing an enzymatically inactive mutant of the FtsH protease (FtsH E415A) or in cells lacking FtsH altogether (DftsH). Approximately 44% of s 32 fractionated to the membrane in a DftsH strain, and this fraction was increased to ,58% in the FtsH E415A strain, raising the possibility that FtsH itself may participate in retention of s 32 at the IM. As the b9 subunit of RNA polymerase, a known interaction partner of s 32 , also fractionated with the membrane, we next tested whether s 32 association with the IM was dependent on its association with RNA polymerase. To this end, we used s 32 D21aa, which is defective in interacting with RNA polymerase [32]. We confirmed that s 32 D21aa did not detectably interact with RNA polymerase ( Figure S1A,B). Yet endogenous WTs 32 and ectopically expressed s 32 D21aa fractionated equivalently to the IM both in DftsH cells (,39%) and in FtsH E415A cells (,58%) ( Figure S2), indicating that s 32 transited to the membrane independent of RNA polymerase.
We next tested whether the pftsY::Tn5 mutation or the homeostatic control region mutation of s 32 disrupted membrane partitioning of s 32 . Both WTs 32 and ectopically expressed s 32 D21aa were defective in partitioning to the IM in pftsY::Tn5 cells ( Figure 5). To look at the effect of disrupting the homeostatic control region on membrane fractionation, we expressed I54Ns 32 as a s 32 D21aa variant (I54Ns 32 D21aa). The size difference allowed us to compare I54Ns 32 D21aa and WTs 32 in the same cells ( Figure S2). Whereas WTs 32 exhibited normal fractionation, I54Ns 32 D21aa showed a severe localization defect, comparable to that of pftsY::Tn5 cells ( Figure 5). We conclude that s 32 targeting to the IM is dependent on both SRP/SR and the s 32 homeostatic control region.

Both SecA and SecY Are Important for Membrane Association of s 32
SecA is an ATP-fueled motor protein that recognizes signal peptides, drives the translocation of secreted proteins through the Sec translocon [33][34][35][36][37], and collaborates with the SRP/SR for integration of a subset of IM proteins into the membrane [33,38]. We previously found that s 32 activity is increased in a SecA(ts) strain [39]. This observation motivated us to explore the relationship of partially digested by endopeptidase V8, was resolved on a 10% SDS-PAGE gel, transferred to nitrocellulose, and incubated with s 32 . The Coomassiestained gel (left) and the nitrocellulose membrane, containing transferred Ffh fragments, probed against s 32  SecA to IM trafficking of s 32 . Indeed, using a SecA(ts) mutant with general defects in protein export (SecAL43P) [40,41], we observed that cells displayed a significant defect in membrane localization of s 32 ( Figure 5), as well as increased s 32 activity ( [39] and unpublished data). In addition, purified SecA, resolved on SDS-PAGE and transferred to nitrocellulose, showed binding affinity for s 32 , suggesting that these two proteins interact ( Figure S3). We conclude that SecA participates in trafficking of s 32 to the IM. SecY forms the core of the SecYEG IM translocon. This multidomain protein has a large cytoplasmic domain (C5) that functionally interacts with SR [42], SecA, and the ribosome [43][44][45][46][47][48][49][50] (Figure 6A). We tested whether 10 previously described secY mutations located in various domains of SecY ( Figure 6A) [51] perturb chaperone-mediated control of s 32 activity and trafficking of s 32 to the IM ( Figure 6B). All mutants had enhanced s 32 activity. This result was not surprising as secY mutants are expected to accumulate secretory protein precursors that titrate chaperones [52]. Importantly, four mutants (secY124, secY351, secY40, secY129) were also defective in chaperone-mediated control of s 32 activity ( Figure 6B), as indicated by a lack of down-regulation of s 32 activity in response to overexpression of one or both of the chaperone systems. We examined the secY351 mutant, which had both high s 32 activity and a significant defect in chaperonemediated inactivation, and found it to be defective in IM trafficking of s 32 ( Figure 5). secY40 and secY351 affect domain C5 ( Figure 6A), implicated in the interaction of SecY with SR, raising the possibility that this interaction is important for both homeostatic control and IM targeting of s 32 .

An Independent Methodology Indicates Association of s 32 with the IM
Alkaline phosphatase is active only in the periplasm, where it forms the disulfide bonds necessary for its activity. Therefore, translational fusions to alkaline phosphatase (PhoA) lacking its own export signal are commonly used as an indicator of membrane targeting by the appended N-terminal sequence [53]. If the appended N-terminal sequence has either an export or insertion sequence, the fusion protein will exhibit alkaline phosphatase activity in vivo because it is partly transported to the periplasmic side of the membrane through the SecYEG translocon. Although s 32 has neither a membrane insertion nor an export sequence, it may contain a sequence that targets it to the cytosolic face of the IM. There is some evidence that the secretory apparatus can recognize the mature domains of exported proteins at low efficiency [54]. If so, proximity of PhoA to the translocon resulting from the IM targeting signal might enable transit of some fraction of PhoA to localize to the periplasmic side of the membrane, where it is active. By random insertion of the transposon probe TnphoA into rpoH, encoding s 32 (see Materials and Methods), we found that a phoA fusion to the first 52 amino acids of s 32 (N52-s 32 -PhoA) showed ,10-fold greater PhoA activity than signal-less PhoA itself, indicating that the Nterminus of s 32 facilitates PhoA export (Table 2). Moreover, PhoA activity enhancement is dependent both on the SRP/SR-dependent trafficking system and on SecY, as both pftsY::Tn5 and secY351 decreased the PhoA activity ,2-fold, whereas leaderless PhoA exhibited little response to these perturbations (Table 2). Thus, this assay is consistent with the idea that the N-terminus of s 32 carries an IM-trafficking sequence and that the targeting process is dependent on SRP and SecY.

Membrane-Tethering of Otherwise Deregulated s 32 Restores Homeostatic Control
The I54Ns 32 mutant and mutants in the IM-targeting machinery (pftsY::Tn5, secA(ts), secY351) were both defective in proper regulation of s 32 and in s 32 association with the IM. This convergence motivated us to test whether artificially tethering s 32 to the IM could restore homeostatic control. To this end, we exploited the bacteriophage Pf3 coat protein. With the addition of three leucine residues in its membrane-spanning region, 3L-Pf3 translocates spontaneously in an orientation-specific manner to the IM, where it inserts in an N-out/C-in orientation [55]. We modified rpoH (encoding s 32 ) at its chromosomal locus to encode a s 32 variant with the 3L-Pf3 membrane-insertion signal attached to its N-terminus (schematized in Figure S4A). Strains carrying   Figure S4B). Thus, s 32 functions when it is tethered to the IM.
We determined whether IM-WTs 32 was subject to homeostatic control circuitry exhibited by WTs 32 . s 32 is maintained at a low level by FtsH degradation, and its activity is decreased by chaperone-mediated inactivation. Both phenotypes are evident by comparing the amount and activity of s 32 in a WT versus a DftsH strain. In a DftsH strain, the level of WTs 32 increases ,30fold because the major protease degrading s 32 is removed (Table 3; Figure S5 [compare lanes 1 and 3]; and [25]). However, the activity of s 32 increases only 3-fold as a consequence of chaperone-mediated activity control, leading to a 10-fold reduction in the S.A. of s 32 in DftsH cells relative to that in WT cells (Table 3 and [56]). Both the level and S.A. of WTs 32 and IM-WTs 32 were closely similar in a DftsH strain, indicating that the chaperone-mediated activity control circuit is active in IM-WTs 32 (Table 3 and Figure S5 [compare lanes 3 and 4]). Additionally, the level of IM-WTs 32 was significantly lower in ftsH + than in a DftsH strain, indicating that IM-WTs 32 was efficiently degraded by FtsH (Table 3 and Figure S5 [compare lanes 2 and 4]). The presence of a contaminating band prevented absolute quantification of IM-WTs 32 levels via Western blot analysis ( Figure S5). However, if the relative S.A. of IM-WTs 32 and WTs 32 are equivalent in the ftsH + strain as we found in the DftsH strain, then the 2-fold decrease in activity of IM-WTs 32 relative to WTs 32 implies a slight increase in the rate of degradation of IM-WTs 32 relative to WTs 32 . Note that the 3L-Pf3 membrane-insertion tag itself is not a signal for FtsH degradation, as the stability of the FliA s factor, which is closely related to s 32 , was unchanged when expressed as  [51]. The region that interacts with FtsY (Domain C5) is boxed in green. (B) Mutations in secY show higher s 32 activity and affect chaperone-mediated activity control of s 32 . The activity of s 32 was measured in WT and secY mutant cells growing at 30uC in LB medium (column 1) or in the same cells following induction of DnaK/J (column 2) or GroEL/S (column 3). Activity is calculated as the differential rate of b-galactosidase synthesis from a chromosomal P tpG -lacZ reporter in each cell type relative to that of WT cells.  Figure S6). In summary, both the chaperonemediated activity control circuit and the FtsH-mediated degradation control circuit are active on IM-tethered s 32 .
Next, we asked whether the forced and stable tethering of s 32 to the IM bypassed the regulatory defects of I54Ns 32 and the reduced-level SR mutant pftsY:::Tn5. I54Ns 32 is degraded poorly by FtsH as its level was 11-fold higher than that of WTs 32 (Table 3; Figure S5 [compare lanes 1 and 6] and [25]). I54Ns 32 also had compromised chaperone-mediated activity control as the high chaperone levels in this strain did not reduce the S.A. of I54Ns 32 (Table 3; and [25]). In stark contrast, both degradation and activity control were restored when I54Ns 32 was converted to IM-I54Ns 32 . FtsH efficiently degraded the membrane-tethered variant: IM-I54Ns 32 was undetectable in ftsH + cells but present at a high level in DftsH cells (Table 3 and Figure S5 [compare lanes 5 and 7]). Additionally, IM-I54Ns 32 and IM-WTs 32 exhibited comparable reductions in relative S.A. of s 32 in DftsH cells (Table 3). Stable tethering of s 32 to the IM also bypassed the regulatory defects of pftsY::Tn5 as IM-WTs 32 in the reduced-level SR background was degraded and subject to chaperone-mediated activity control. Indeed, IM-WTs 32 behaved identically in WT and pftsY::Tn5 strains, exhibiting comparable s 32 activity at a protein level below detection (Table 3 and Figure S5 [compare lanes 8 and 9]). Finally, IM-tethering relieved the growth defects of both I54Ns 32 ( Figure S7A and C) and of pftsY::Tn5 ( Figure S7B, C, and D). In summary, stable tethering of s 32 to the IM restored both homeostatic control and normal growth to cells with a defective s 32 homeostatic control region and to cells with a compromised SRP/SR co-translational targeting apparatus.

Discussion
Our work has led to a revised model of the HSR circuitry ( Figure 1B). s 32 first transits to the IM via an SRP/SR-dependent process and is then subjected to the chaperone-mediated activity control and FtsH-mediated degradation control that have been previously described. This revised model enables the homeostatic control circuit to integrate information on both cytosolic and IM status. Importantly, the efficiency of co-translational protein targeting depends on the cumulative effect of multiple SRP checkpoints including differences in cargo binding affinities, kinetics of SRP-SR complex assembly, and GTP hydrolysis [57]. Multiple checkpoints and the fact that SRP is sub-stoichiometric relative to translating ribosomes (,1:100; SRP molecules to translating ribosomes [58]) may allow SRP to modulate the extent of IM-localization of s 32 during times of stress and/or increased protein flux. Thus, s 32 down-regulation through its localization to the membrane could be alleviated when the IM is disturbed or SRP is overloaded in assisting membrane protein biogenesis. This feed-forward mechanism allows the s 32 homeostatic control to sense the state of cytosolic and IM proteostasis before unfolded proteins accumulate to a significant extent. Interestingly, ffh (encoding the protein subunit of the SRP) is a s 32 regulon member as its expression increases at least 3-fold following induction of s 32 either by heat shock or by deletion of dnaK/J ( [30] and unpublished data). This could provide an additional connection between s 32 and protein flux to the IM. Finally, and more speculatively, given the demonstrated involvement of SecA in IM targeting of s 32 and its direct interaction with s 32 , the s 32 homeostatic control circuit may also monitor protein flux through SecA to the periplasm and outer membrane.
The idea that the high activity of s 32 in the I54Ns 32 homeostatic control mutant and in SRP/SR mutants (eg. pftsY::Tn5) results from s 32 mislocalization to the cytosol and consequent homeostatic dysregulation, rather than from chaperone titration by a buildup of unfolded proteins, is supported by our data. First, forced IM-tethering overcomes the inviability of the I54Ns 32 mutation in the DftsH strain background (Table 3), as well as the growth defects of I54Ns 32 and pftsY::Tn5 ( Figure S7), suggesting that high expression of s 32 is aberrant and deleterious to cells, rather than required to remodel misfolded proteins. This is reminiscent of previous findings that reduced-function s 32 mutants suppress physiological defects of a DdnaK strain [59] and that overexpression of HSPs was deleterious to growth [13,60]. Second, secY mutants dysregulated in chaperone-mediated activity control were not distinguished by their extent of s 32 induction. This is contrary to the prediction of the chaperone titration model, which posits that secY mutants with the highest s 32 induction would have the highest level of unfolded proteins. These mutants would then be refractory to activity control because the additional chaperones resulting from chaperone overexpression would actually be needed to remodel the misfolded protein burden. We conclude that homeostatic dysregulation of s 32 results from s 32 mislocalization, rather than from the buildup of unfolded proteins.  The molecular nature of IM-localized s 32 remains unclear. Prediction programs [61,62] do not detect either a signal peptidelike or transmembrane sequence in s 32 . We favor the idea that following transit to the IM, s 32 is maintained at the membrane via interactions with other proteins and/or lipid head groups during its short half-life in the cell . Indeed, we have already demonstrated interactions between s 32 and several membraneassociated or IM proteins, including SRP, SecA, and FtsH itself. Moreover, the chaperone systems regulating s 32 (DnaK/J/GrpE and GroEL/S) show partial distribution to the membrane [63][64][65][66][67][68], whereas other potential membrane-associated protein partners have not yet been tested for s 32 interaction (e.g., SecY and additional members of the Sec machinery). Each of these proteins could result in partial membrane localization of s 32 , as was shown for FtsH where deletion of the protein decreased localization relative to cells with the protease-dead mutation FtsH E415A. Importantly, if s 32 is membrane associated via transient protein-protein and/or proteinlipid interactions, some s 32 may dissociate from the membrane during cell lysis, as was demonstrated for FtsY, another peripheral membrane protein [69,70]. Therefore, although we report that ,50% of s 32 is membrane-associated, the fraction of s 32 that is actually IM-localized may be significantly higher.
IM-associated s 32 may provide regulatory flexibility not possible for IM-tethered s 32 . For example, during times of high stress, s 32 may be able to dissociate from the membrane to escape homeostatic control. These excursions could be transient if SRP were able to transport s 32 posttranslationally, a possibility suggested by the fact that full-length, fully folded s 32 binds to SRP (Figures 2 and 3 and Figure S1). Additionally, IM-tethered s 32 is more rapidly degraded than IM-associated s 32 , suggesting that tethering makes s 32 a better FtsH substrate. This could diminish the ability of the cell to regulate the rate at which FtsH degrades s 32 , which is of physiological significance during temperature upshift [8]. The transient reduction in s 32 degradation following increased temperature contributes significantly to the rapid build-up of s 32 during heat shock [8].
Membrane localization is widely used to control s factors [71,72]. The inactive B. subtilis SigK pro-protein is membrane inserted; cleavage of its N-terminal pro-sequence releases SigK [73,74]. Cleavage is coordinated with passage of a checkpoint in spore development to provide just-in-time SigK activity [75]. Additionally, many s factors are held in an inactive state at the membrane by cognate membrane-spanning anti-s factors and released as transcriptionally active proteins when stress signals lead to degradation of their anti-s [71,76]. IM-localization of s 32 serves a conceptually distinct role as s 32 is equally active in the cytoplasm or at the IM. Instead, the localization process itself is the key regulatory step in two ways: localization is both regulated by protein folding status and is prerequisite for proper function of the homeostatic control circuit.
The SRP-SR co-translational targeting system has an important role in maintaining proteostasis. SRP-SR minimizes aggregation and misfolding of the approximately 20%-30% of proteins destined for the IM, by making their translation coincident with membrane insertion. Our finding, that SRP/SR-mediated transit of s 32 to the IM is also critical for proper control of the HSR, points to a significant new regulatory role for the co-translational targeting apparatus in protein-folding homeostasis. This finding also raises important mechanistic questions. Our in vitro interaction results suggest a direct, but weak, interaction between full-length s 32 and the M-domain of SRP. The prevailing paradigm suggests that the M-domain interacts only with nascent polypeptides with particularly hydrophobic signal sequences. It is possible that s 32 is detected co-translationally, as the Region 2.1 N-terminal a-helical structure, which resembles a hydrophobic signal sequence, may be recognized by the SRP. Alternatively, we note that the SRP chloroplast homolog (cpSRP54) has a dedicated posttranslational targeting mechanism for several fully translated membrane proteins [77], and E. coli SRP, alone or in combination with additional accessory factors (e.g., other s 32 interactors, such as chaperones or SecA), may target mature s 32 to the membrane in vivo. It remains to be determined whether an interaction between full-length s 32 and SRP, or a novel co-translational targeting interaction by the SRP-SR system, mediates transit of s 32 to the membrane.

Isolation of pftsY::Tn5 Mutant
Strain CAG48275 [25], which is DlacX74, contains the prophage JW2 (P htpG -lacZ), and a chromosomal dnaK/J locus driven from P A1/lacO-1 under control of lacI q [14] was grown in LB, induced with 1 mM IPTG to overexpress DnaK/J chaperones, treated with Tn5, and plated at 30uC on X-gal indicator plates containing kanamycin to select for strains containing Tn5. Blue colonies were picked and tested for higher s 32 activity and for feedback resistance to excess DnaK/J [25]. Tn5 insertion sites were determined by DNA sequencing.

b-Galactosidase Assay
Overnight cultures (LB medium) were diluted 250-fold and grown to exponential phase (OD 600 = 0.05-0.5). Samples were taken at intervals starting at OD 600 = 0.05, and s 32 activity was monitored by measuring b-galactosidase activity expressed from the s 32 -dependent htpG promoter, as done previously [25].

In Vivo Co-Immunoprecipitations
Cells were grown to OD 600 ,0.35 in LB medium at 30uC, harvested, washed two times with 16 PBS, resuspended in Lysis Buffer (20 mM Hepes-KOH, 150 mM NaCl, 10 mM EDTA, 10% glycerol, pH 7.5), and lysed by passing 46 through an Avestin EmulsiFlex-C5 cell homogenizer at 15,000 psi. Cellular debris was spun out and the supernatants were incubated with anti-Ffh or anti-FtsY antibodies at 4uC for 14 h by rotation. TrueBlot anti-Rabbit Ig IP Beads (eBioscience) were added and the supernatants rotated for an additional 2 h at 4uC. Immunocomplexes were isolated by centrifugation and washed 56 in Lysis Buffer without EDTA, and eluted in TCA Resuspension Buffer (100 mM Tris (pH 11.0), 3% SDS) containing LDS Sample Buffer (Invitrogen). Proteins were separated by 10% SDS-PAGE, analyzed by immunoblotting using anti-s 70 and anti-s 32 antibodies, and imaged using fluorescent secondary antibodies (as described below).

Identification of Direct Protein-Protein/Domain Interactions
Detection of a direct protein-protein/domain interaction was carried out exactly as previously described [83]. Proteins were separated on 10% SDS-PAGE. Partially proteolyzed Ffh was obtained by incubating 400 mg of purified Ffh with 4 mg of Glu-C endopeptidase (New England Biolabs) at 25uC in 10 mM Na-HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT, 10 mM MgCl 2 , and 10% glycerol. An aliquot of the reaction was taken out at various times (0, 5, 10, 15, 30, 45, 60, 120, 180, and 330 min) and stopped by addition of 56 volume of 56 SDS-sample loading buffer. The samples were then analyzed by blot overlay with s 32 as the probe.

In Vivo Crosslinking, 66HIS-tag Affinity Isolation and Co-Immunoprecipitation
In vivo crosslinking experiments were carried out essentially as described previously [84]. Strains of CAG48238 carrying pEVOL-pBpF were further transformed with pRM5 or pRM17. Cells were grown at 30uC in L medium containing 0.02% arabinose and 1 mM pBPA, induced with 1 mM IPTG for 1 h, and UV-irradiated for 0 or 10 min at 4uC. For analysis of whole cell samples, total cellular proteins were precipitated with 5% trichloroacetic acid, solublized in SDS sample buffer, and analyzed by 7.5% SDS-PAGE and immunoblotting.
Co-immunoprecipitations were carried out as follows: UVirradiated cells were suspended in 10 mM Tris-HCl (pH 8.1) and disrupted by sonication at 0uC. After removal of total membranes by ultracentrifugation, proteins were precipitated with 5% trichloroacetic acid, washed with acetone, and solubilized in buffer containing 50 mM TrisHCl (pH 8.1), 1% SDS, 1 mM EDTA. The samples were then diluted 33-fold with NP40 buffer (50 mM TrisHCl (pH 8.1), 150 mM NaCl, 1% NP40). After clarification, supernatants were incubated with anti-Ffh antibodies and TrueBlot anti-Rabbit Ig IP Beads (eBioscience) at 4uC for 13 h with rotation. Immunocomplexes were isolated by centrifugation, washed 2 times with NP40 buffer and then once with 10 mM TrisHCl (pH 8.1), and dissolved in SDS sample buffer. Proteins were separated by 7.5% SDS-PAGE and analyzed by immunoblotting using anti-Ffh and anti-s 32 antibodies, TrueBlot anti-Rabbit IgG (eBioscience), and Can Get Signal immunoreaction enhancer solution (TOYOBO Life Science, Japan).
For 66HIS-tag affinity isolation, UV-irradiated cells were suspended in 10 mM Tris-HCl (pH 8.1) containing 6 M urea and disrupted by sonication at 0uC. After clarification by ultracentrifugation, the soluble fraction was loaded onto the TALON resin (TAKARA BIO, Inc., Japan). After washing the resin with wash buffer (50 mM TrisHCl (pH 7.0), 300 mM KCl, 6 M urea, 20 mM imidazole), bound proteins were eluted with wash buffer containing 300 mM imidazole. Proteins were precipitated with 5% trichloroacetic acid, solublized in SDS sample buffer, and analyzed by 7.5% SDS-PAGE and immunoblotting.

Gel Filtration
Purified proteins were run on a Superdex 200 PC 3.2/30 column, pre-equilibrated with Buffer A (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM MgCl 2 , 2 mM DTT). Purified proteins or protein complexes were run with Buffer A at a flow rate of 40 mL/ min, and collected fractions were analyzed by SDS-PAGE and immunoblotting for s 32 . SRP was formed by incubating purified Ffh with 1.56 molar excess of purified 4.5S RNA on ice for 10 min. To form SRP-s 32 complexes, 3 mM of purified WTs 32 or I54Ns 32 was mixed with 106 molar excess of SRP; proteins were incubated on ice for 30 min before analysis by gel filtration.

Construction of s 32 -PhoA Fusion
A 52-s 32 -Tn5PhoA fusion was initially isolated by random screening for PhoA + clones on PhoA indicator plates-using a strain carrying a TnphoA transposon probe [85] on a low-copy plasmid and P lac -rpoH (encoding s 32 ) on a multicopy plasmid. The fusion used in this article (N52-s 32 -PhoA lacking the transposon but containing the first 52 amino acids of WTs 32 ) was subsequently constructed by standard recombinant DNA techniques. Direct construction of fusions past amino acid 52 of s 32 was very unstable, precluding their analysis.

Cell Fractionation
Cells were grown to OD 600 = 0.3-0.4, harvested, and resuspended in ice-cold Buffer B (10 mM Tris-Acetate (pH 7.4), 10 mM Mg(OAc) 2 , 60 mM NH 4 Cl, 1 mM EDTA, supplemented with 1 mM PMSF) to an OD 600 of 15. Cells were immediately lysed by passaging the extracts through an Avestin EmulsiFlex-C5 cell homogenizer at 15,000 psi, and subjected to low-speed centrifugation to remove cell debris and un-lysed cells. Membranes were collected by ultracentrifugation in an Optima benchtop centrifuge (Beckman-Spinco) with a TLA 100.3 rotor (60 min; 52,000 rpm; 4uC). The supernatant was saved as the soluble fraction, while the pellet was washed 36 with Buffer B and then resuspended in Buffer C (50 mM HEPES-KOH pH 7.6, 50 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5% n-Dodecyl b-D-maltoside, and 5% glycerol). Both the soluble and membrane fractions were precipitated in trichloroacetic acid (13% vol/vol), incubated on ice for 30 min, and then overnight at 4uC. Precipitated proteins were then washed with ice-cold acetone and analyzed by SDS-PAGE and immunoblotted for s 32 (Neoclone), b9 (Neoclone), s 70 (Neoclone), RseA [86], and RuvB (Abcam) with fluorescent secondary antibodies (LI-COR Biosciences) used for detection. The percentage of s 32 in each fraction was determined by direct scanning and analyzing bands with ImageJ software (National Institutes of Health).

RNA Polymerase Pull-Downs
Cells were grown to OD 600 = 0.35-0.45, harvested, and resuspended in ice-cold Buffer D (50 Tris-HCl, pH 8.0, 0.1 mM EDTA, 150 mM NaCl, and 5% glycerol) to an OD 600 of 20. Lysozyme was added to 0.75 mg/mL and cells were incubated on ice for 30 min, followed by sonication, then subjected to low-speed s 32 Interacts with the Signal Recognition Particle centrifugation to remove cell debris and unlysed cells. Lysates were then incubated with pre-equilibrated, pre-blocked (Buffer D containing 5% Bovine Serum Albumin, 0.1 mg/mL dextran) Softag 4 Resin (Neoclone) overnight at 4uC. Bound proteins were washed 36 with Buffer D and eluted with 46 LDS NuPAGE Buffer (Life Technologies). To collect lysates and eluted proteins, 0.05 mM of Strep-66H-tagged s 32 was added as a loading and blotting control during analysis by SDS-PAGE and Western blotting against s 32 .

Construction of 3L-Pf3 Fusion Proteins
The 3L-Pf3 genetic sequence was created by carrying out standard polymerase chain reaction using the following overlapping oligos: 59-atgcaatccgtgattactgatgtgacaggccaactgacagcggtgcaagc-39, 59-taccattggtggtgctattcttctcctgattgttctggccgctgttgtgctggg-39, 59-aaagaattgcgctttgatccagcgaatacccagcacaacagcggccagaa-39, and 59aagaatagcaccaccaatggtagtgatatcagcttgcaccgctgtcagtt-39. The stitched oligos were then cloned using TOPO TA cloning (Invitrogen) and sequenced. To construct chromosomal 3L-Pf3-s 32 , PCR was carried out to stitch the 3L-Pf3 gene sequence flanked by the first 500 base pairs of the s 32 open reading frame and 500 base pairs upstream of the start codon, and subsequently cloned into the pKNG101 suicide vector. The 3L-Pf3 sequence was then integrated 59 and in-frame with the chromosomal rpoH gene by double homologous recombination. Counterselection of sacB on pKNG101 was carried out on 10% sucrose media (5 g/L Yeast Extract, 10 g/L Tryptone, 15 g/L Bacto Agar, 10% sucrose) [25,87]. Clones were sequenced to verify chromosomal integration of the 3L-Pf3 sequence in the correct reading frame.
To construct pTrc99A expressing 3L-Pf3-FliA, flgM and fliA (in that order) were cloned as an operon, with the sequence 59-ccgtctagaattaaagAGGAGaaaggtacc-39 added between the two genes in the vector; the Shine-Dalgarno site is designated in uppercase. Two plasmids were created-one with just flgM and fliA, unmodified, and one where the 3L-Pf3 sequence was cloned 59 to and in-frame with fliA. Clones were sequenced to verify correct sequences and proper reading frame. Expression was from the leaky pTrc promoter, and experiments were only carried out after fresh transformation into the parental CAG48238 strain. Levels of FliA were analyzed by SDS-PAGE and immunoblotting with antibodies against FliA (Abcam).

Immunoblotting
Cells were re-suspended in equal volumes of Buffer C, with the addition of trichloroacetic acid (final 13% vol/vol), kept on ice overnight, and the precipitate collected by centrifugation. Pellets were washed with acetone and resuspended in 16 LDS NuPAGE Buffer (Life Technologies). Serial dilutions of WT and mutant samples were loaded onto a polyacrylamide gel, and proteins transferred to nitrocellulose membranes. The blots were first probed with primary antibodies and then with anti-primary fluorescence-conjugated secondary antibody (Licor). Immunoblots were scanned at the appropriate wavelengths for detection. Fold increase (protein level experiments) was estimated by comparison with a dilution series of samples from the WT strain. Fold decrease after addition of chloramphenicol (protein stability experiments) was determined by direct scanning and analyzing bands with ImageJ software (National Institutes of Health). Figure S1 s 32 D21aa, a C-terminal truncation of s 32 , is defective in binding to RNA polymerase in vivo. (A) Immunoprecipitation of RNA polymerase-bound native s 32 and s 32 D21aa. s 32 D21aa was expressed from pTrc99A in DftsH cells, induced to levels comparable to endogenous s 32 , grown to midexponential at 30uC in LB medium and the amount of s 32 bound to the anti-b9 resin (Softag4; Neoclone) and remaining s 32 in the supernatant was quantified by immunoblotting using a polyclonal antibody against s 32  Strains were grown to OD 600 ,0.35, precipitated by addition of TCA to 13% final (vol/vol). Levels of s 32 and s 32 variants were determined by quantitative immunoblotting (see Materials and Methods). The experiment was carried out $5 times, with an example blot shown. These are the raw data used to obtain level values for s 32 and its variants shown in Table 3. Averaged quantification of the amount b9 served as a loading control, and levels of FtsH and FtsY are additionally shown. The genetic backgrounds of the mutant strains are shown below the blots. The specific protein probed on each blot is shown to the right. Note that IM-s 32 and IM-I54Ns 32 run as a smear, most likely because the membrane localization signal adopts multiple conformations during SDS-PAGE electrophoresis. To minimize this problem, gels were run very slowly (60-80 volts). Amount of IM-s 32 variants was calculated over the entire smear. Additionally, there is a contaminating band in all samples marked with an asterisk (*) that runs approximately at the same molecular weight as IM-s 32 . This contaminating band prevents accurate quantification of samples with low amounts of IM-s 32 (lanes 2, 7, and 9).