INTRODUCTION

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The signal recognition particle (SRP) was first identified in mammalian cells as a soluble factor that is required for the entry of virtually all proteins into the secretory pathway. Mammalian SRP, a ribonucleoprotein complex composed of six polypeptides and a 300-nucleotide RNA, guides or "targets" nascent polypeptides to transport sites in the endoplasmic reticulum (ER) (reviewed in reference 52). The SRP 54-kDa subunit (SRP54) specifically binds to the N-terminal signal sequences of secreted proteins (23, 25) and the transmembrane (TM) domains of integral membrane proteins (18) as they emerge from translating ribosomes and then releases them after interaction with a heterodimeric receptor in the ER membrane (11, 46). Release of nascent chains appears to be coupled to their insertion into a transport channel or "translocon" (7, 12, 17), the core of which is a heterotrimer called the Sec61p complex (13).

Although homologs of SRP and its receptor have been identified in every organism that has been examined and have been shown to target proteins to both the yeast ER (14, 34) and the bacterial inner membrane (8, 45, 48), it is clear that unicellular organisms also possess alternative targeting pathways (15, 24, 43, 53). In Escherichia coli, SRP is comprised solely of an SRP54 homolog (Ffh) and a small RNA (4.5S RNA) (37, 41), and the SRP receptor consists of a single subunit (FtsY) (1, 42). To date, these factors have been shown to be required only for the efficient insertion of polytopic inner membrane proteins (IMPs) into the inner membrane (8, 45, 48). Most or all exported proteins can be targeted to transport sites by chaperones such as SecB that simply keep their passengers in a loosely folded, transport-competent conformation. Chaperones differ from SRP in that they bind in a relatively promiscuous fashion to sequences within the mature region of presecretory proteins (6, 10) and in that they appear to function at least in part in a posttranslational fashion (26). Despite the multiplicity of targeting pathways in yeasts and bacteria, essentially all proteins are thought to be transported across the membrane by a single translocon (reviewed in reference 40). Together with the peripheral membrane protein SecA, the SecY complex, which is closely related to the Sec61p complex (12, 16), forms the core of the translocon in prokaryotes.

The features of an IMP that require utilization of the SRP targeting pathway for efficient insertion have not yet been identified. Several studies have suggested that bacterial SRP, like eukaryotic SRP (51), recognizes proteins on the basis of hydrophobicity. Experiments in which the interaction of E. coli SRP with model nascent chains has been assessed by UV-cross-linking and preprotein translocation assays (38, 49, 50) have demonstrated a correlation between signal sequence hydrophobicity and SRP binding in vitro. These studies, together with recent in vivo experiments on the targeting of an M13 procoat derivative (H1-procoat) that has an unusually hydrophobic signal sequence (9), imply that the presence of a highly hydrophobic segment is sufficient to route a protein into the SRP targeting pathway. These experiments do not examine, however, whether the hydrophobicity of IMPs is the distinguishing feature that necessitates targeting by SRP for efficient insertion. Indeed, the observation that the insertion of some IMPs is not detectably affected by disruption of the SRP pathway (48) suggests that certain proteins that are targeted by SRP under normal growth conditions can also utilize alternative targeting pathways effectively. All of the IMPs that have been shown to require SRP for efficient membrane insertion are complex proteins that contain multiple transmembrane (TM) domains (8, 48) or, in the case of H1-procoat, one TM domain and an unusually long and hydrophobic signal sequence (9). Thus, it is unclear whether the SRP requirement is due to the presence of multiple TM domains, key individual TM domains, or hydrophilic domains that lie outside the membrane.

In this report we describe experiments designed to identify the features of an E. coli IMP that obligate utilization of the SRP targeting pathway. To simplify interpretation of the experiments, we examined the targeting requirements of model bitopic proteins that contain only a single TM domain. These proteins differ from exported proteins in two important respects. First, their TM domain, which serves as a targeting signal, is longer and much more hydrophobic than most signal peptides. Second, they contain cytoplasmic and periplasmic segments that may be structurally distinct from the mature domains of exported proteins. We found that the biogenesis of only some bitopic proteins was significantly affected by disruption of the SRP targeting pathway. Genetic engineering of one of the affected proteins (an AcrB derivative) revealed that the SRP requirement for efficient insertion is dictated by the periplasmic domain and not by the TM domain. Furthermore, we found that different methods of disrupting the SRP pathway, including exhaustive SRP depletion, only partially blocked the insertion of both bitopic and polytopic IMPs. Considered together, these results show that a variety of factors, in addition to hydrophobicity, can contribute to SRP dependence and suggest that the SRP pathway facilitates, but is not absolutely required for, the insertion of many IMPs.

	MATERIALS AND METHODS

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Reagents, media, and bacterial manipulations. E. coli MC4100 [F araD139 (argF-lac)U169 rpsL150 relA1 thi fib5301 deoC1 ptsF25 rbsR] and WAM113 [MC4100 ara⁺ ffh::kan-1 (Para-ffh Ap^r)] have been described previously (4, 36). Medium preparation and basic bacterial manipulations were performed by standard methods (31). Selective media contained 100 µg of ampicillin or 40 µg of chloramphenicol per ml. Indicator plates for alkaline phosphatase (AP) activity contained 40 µg of 5-bromo-4-chloro-3-indolylphosphate (BCIP; Boehringer Mannheim) per ml. Taq polymerase (AmpliTaq; Perkin-Elmer) was used to amplify DNA fragments by PCR. The antiserum to AP was obtained from 5 Prime3 Prime.

Plasmid construction. All AP fusions used in this study were subcloned into pACYC184 or its derivative pNU74 (48). The construction of AP fusions to AcrB at amino acid residue 576 (AcrB 576-AP) and to Tsr at amino acid residue 164 (Tsr 164-AP) has been described (29, 48). The bitopic AcrB 265-AP fusion was constructed by first digesting plasmid S215 (48) with NruI and SalI to delete most of AcrB. The resulting plasmid was then cut with SalI and ligated to a BsiHKAI-XhoI fragment of plasmid pHI-1 (20) containing the AP gene by using the oligonucleotide adapters 5'-TCGAAGGCGATATTACTGCACCCGGCGGTGCT-3' and 5'-CCGCCGGGTGCAGTAATATCGCCT-3' to create plasmid pJN4. A fragment containing the fusion was isolated by digesting pJN4 with SmaI and HindIII and was ligated to pACYC184 digested with NruI and HindIII to create pJN6. The YfgA 139-AP fusion was constructed by using plasmid S356 (48), which contains the YfgA gene embedded in a fragment of genomic DNA (GenBank accession number ECAE000338; bp 1301 to 2939). S356 was digested with BlpI and SalI and ligated to the AP-containing fragment of pHI-1 described above by using the oligonucleotide adapters 5'-TCAGCAGGGCGATATTACTGCACCCGGCGGTGCT-3' and 5'-CCGCCGGGTGCAGTAATATCGCCCTGC-3'. The MdoH 173-AP fusion was constructed from plasmid E140 (48) by random TnphoA insertion (28).

Pulse-chase labeling, immunoprecipitation, and immunoblots. For experiments in which a dominant lethal ftsY allele was overexpressed, MC4100 transformed with pTRC-ftsY(G385A) (48) and a plasmid encoding an AP fusion were grown to an optical density at 550 nm (OD₅₅₀) of 0.4 in M9 medium containing 0.4% glucose and all L-amino acids except methionine and cysteine. For experiments in which Ffh was depleted, WAM113 cells transformed with a plasmid encoding AcrB 576-AP were diluted from an overnight culture to an OD₅₅₀ of 0.005 in M9 medium supplemented with 0.2% fructose, amino acids, and either 0.2% arabinose or 0.2% glucose and then grown to log phase. At appropriate times, aliquots of cells were removed and labeled with Tran³⁵S-label (ICN), spheroplasted, treated with proteinase K, and trichloroacetic acid (TCA) precipitated as described previously (48).

	RESULTS

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SRP is required for the efficient insertion of a subset of bitopic IMPs. To determine whether the presence of multiple TM domains dictates SRP dependence, we analyzed the membrane insertion of a variety of single-TM-domain-containing proteins (bitopic proteins) after disruption of the SRP pathway. All of the bitopic proteins examined have a type II (N terminus inside) orientation. To conduct these experiments, we truncated several polytopic proteins and one naturally occurring bitopic protein (YfgA, a protein of unknown function) and constructed in-frame periplasmic fusions to AP. We then analyzed the insertion of the fusion proteins by using a simple protease protection assay in which exogenously added protease releases the AP domains of properly inserted proteins but is unable to digest fusion proteins remaining in the cytoplasm (47). Plasmids encoding the AP fusion proteins were introduced into MC4100, and the cells were grown to mid-log phase. The function of the SRP pathway was then disrupted by adding isopropyl--D-thiogalactopyranoside (IPTG) to induce the expression of a dominant lethal ftsY allele (G385A). Previous studies have shown that synthesis of the mutant FtsY protein blocks the activity of the SRP pathway more rapidly than Ffh depletion and is equally effective (48). After expression of the ftsY(G385A) allele was induced, cells were labeled with radioactive amino acids. The insertion of newly synthesized AP fusion proteins was then analyzed by treating spheroplasted cells with protease and immunoprecipitating AP-containing polypeptides.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Insertion of a subset of bitopic IMPs is inhibited by overexpression of ftsY(G385A). MC4100 cells transformed with pTRC-ftsY(G385A) and a second plasmid encoding an AP fusion were grown to mid-log phase. The cultures were then divided in half; one portion was left untreated (lanes 1 and 2), and IPTG was added to the other portion (lanes 3 and 4) to induce ftsY(G385A) overexpression. The insertion of AcrB 265-AP (A), Tsr 164-AP (B), YfgA 139-AP (C), and MdoH 173-AP (D) was analyzed 40 min after IPTG addition by pulse-chase labeling and immunoprecipitation of AP-containing polypeptides from protease-treated spheroplasts as described in Materials and Methods. The length of the chase is indicated. The diagrams illustrate the structure of each IMP. In all cases, the N terminus is located in the cytoplasm, and the C terminus is in the periplasm.

SRP dependence is conferred by the periplasmic domain of AcrB 265-AP. In comparing the sequences of the bitopic fusion proteins in the experiments described above, we noticed a correlation between the hydrophobicity of the TM domains and the dependence on SRP for efficient insertion. As measured on the GES scale by the TopPred II computer application set at the default parameters (5), the hydrophobicity scores of the predicted TM domains of AcrB 265-AP and YfgA 139-AP were 2.27 and 2.35, respectively, whereas the TM domains of Tsr 164-AP and MdoH 173-AP scored only 1.88 and 1.27, respectively. To test whether the SRP dependence of AcrB 265-AP biogenesis is due to the extreme hydrophobicity of its TM domain, we replaced the native membrane anchor with the TM domain of Tsr 164-AP. We first engineered restriction sites into the plasmid encoding AcrB 265-AP to facilitate removal of the TM segment. In order to create these sites, it was necessary to introduce a leucine-to-arginine mutation at amino acid residue 30 immediately following the TM domain. Subsequently, the segment encoding the TM domain of the resulting construct (AcrB* 265-AP) was excised and replaced with oligonucleotides encoding the TM domain of Tsr 164-AP to create AcrB* 265-AP (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Summary of the AcrB 265-AP derivatives used in this study. The AcrB 265-AP protein (top line) contains a TM domain (amino acid residues 9 to 29 [solid rectangle]) and an AP moiety fused at residue 265 (shaded rectangle). The construction of all derivatives is described in Materials and Methods. AcrB* 265-AP (second line) contains a leucine-to-arginine point mutation () at residue 30, and AcrB* 265-AP (third line) contains both the L30R mutation and the first TM domain of Tsr (hatched rectangle) in place of the native AcrB TM domain. The location of deletions in seven additional derivatives (1-7) is indicated by dashed lines. The effect of the disruption of the SRP pathway on the insertion of each derivative is indicated. +, Defect observed; , defect not observed; N.A., not testable.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Insertion of an AcrB 265-AP derivative containing a Tsr TM domain is inhibited by overexpression of ftsY(G385A). The insertion of AcrB 265-AP (A), AcrB* 265-AP (B), AcrB* 265-AP (C), and AcrB 2651-AP (D) in MC4100 was analyzed as described in the legend to Fig. 1. IPTG was added to the cells in lanes 3 and 4 to induce ftsY(G385A) overexpression. The length of the chase is indicated. The position of the point mutation in AcrB* 265-AP and AcrB* 265-AP is indicated (), and each distinct TM domain is represented by different shading.

View larger version (38K): [in this window] [in a new window]   FIG. 4.   Large deletions in the AcrB 265-AP periplasmic domain abolish SRP dependence. The insertion of AcrB 265-AP (A), AcrB 2652-AP (B), AcrB 2653-AP (C), AcrB 2654-AP (D), AcrB 2655-AP (E), AcrB 2656-AP (F), and AcrB 2657-AP (G) in MC4100 was analyzed as described in the legend to Fig. 1. The deletions in each plasmid are summarized in Fig. 2. IPTG was added to the cells in lanes 3 and 4 to induce ftsY(G385A) overexpression. The length of the chase is indicated.

Exhaustive SRP depletion does not completely abolish the insertion of a polytopic IMP. In all experiments that we performed with bitopic AcrB-AP and YfgA-AP fusions, overexpression of the ftsY(G385A) allele produced only partial insertion defects. In all previous studies, however, disruption of the SRP targeting pathway only partially blocked the insertion of polytopic IMPs as well (8, 9, 48). Although different methods have been used to eliminate SRP activity, each method has produced similar effects on IMP insertion. These results raise the possibility that the biogenesis of even extremely hydrophobic polytopic IMPs is not entirely SRP dependent. To ensure that these intermediate effects were not due to an incomplete disruption of the SRP pathway, we examined the insertion of the polytopic AcrB 576-AP fusion under extreme conditions in which virtually no SRP remained in the cells.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Exhaustive depletion of SRP does not completely block the insertion of a polytopic IMP-AP fusion. (A) Growth of WAM113 transformed with a plasmid expressing AcrB 576-AP in the presence of arabinose () or glucose (). (B) Cells grown in the presence of arabinose or glucose in the experiment depicted in panel A were removed from cultures at the indicated time point, and the Ffh levels were measured by Western blot. The amount of Ffh in cells grown in glucose is expressed as a percentage of the amount of Ffh in cells grown in arabinose at the indicated time point. (C) The insertion of AcrB 576-AP was examined in the experiment depicted in panel A after 7 h of Ffh depletion (arrow) by pulse-chase labeling and immunoprecipitation of AP-containing polypeptides from protease-treated spheroplasts as described in Materials and Methods. Lanes: 1 to 3, cells grown in arabinose; 4 to 6, cells grown in glucose. The length of the chase is indicated.

	DISCUSSION

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In this report we provide evidence that a combination of structural features, rather than the mere presence of one or more TM domains, determines whether the efficient biogenesis of an E. coli IMP is dependent on the SRP targeting pathway. This conclusion emerged from experiments in which the fate of model IMP-AP fusions was examined after severe inhibition of the SRP pathway. We found that the efficient membrane insertion of some, but not all, bitopic IMPs required SRP activity. Whereas these results demonstrate that the presence of multiple TM domains is not a prerequisite for SRP dependence, they also show that the presence of a single highly hydrophobic targeting signal is not sufficient to require utilization of the SRP pathway. Detailed analysis of a model bitopic protein revealed that SRP dependence was due to the presence of a periplasmic domain. Furthermore, we observed that the insertion of a highly hydrophobic protein that contains seven TM domains was only partially blocked even after extensive SRP depletion. Taken together, these results suggest the existence of an alternative targeting pathway and imply that hydrophobicity is only one of a number of factors that determine the degree to which a protein is required to utilize the SRP targeting pathway for efficient insertion into the membrane. Although all of our experiments were performed with IMP-AP fusions, the observation that the insertion of an IMP that resembles a native protein is also only partially SRP dependent (9) suggests that access to SRP-independent targeting pathways was not due to the presence of an AP domain. Nevertheless, it will be important to confirm our results with IMPs that lack a large globular domain derived from an exported protein.

The observation that major determinants of SRP dependence reside in hydrophilic segments of IMPs leads to the unexpected conclusion that the biogenesis of at least some SRP substrates can be rescued by alternative targeting pathways in the absence of SRP. Although the experiments presented here only address the consequences of eliminating the SRP pathway, numerous studies on mammalian (51), yeast (30, 33), chloroplast (19), and bacterial (9, 38, 49, 50) SRPs strongly suggest that SRP binds to nascent polypeptide chains that exceed a threshold hydrophobicity. These studies predict that the hydrophobicity of TM domains should route most, if not all, E. coli IMPs into the SRP pathway. Whereas under normal physiological conditions it is possible that SRP provides the primary targeting pathway for IMPs by binding to TM domains as they emerge during translation, our results indicate that disruption of the SRP targeting pathway affects the biogenesis of only a subset of these proteins. By contrast, it has been shown in yeast cells that there is a correspondence between those proteins that are most likely to be targeted by SRP (namely, those that have particularly hydrophobic signal sequences) and those whose translocation is most SRP dependent (33). Thus, the SRP dependence of E. coli proteins, unlike eukaryotic proteins, cannot be predicted solely on the basis of the primary amino acid sequence.

Clues as to why SRP is required for the targeting of bitopic proteins such as YfgA 139-AP and AcrB 265-AP emerge from a consideration of the mechanism by which a periplasmic or cytoplasmic domain might affect membrane insertion. The simplest interpretation of the data is that, in the absence of SRP, the hydrophilic domains of these proteins either promote folding into an insertion-incompetent conformation or mask the TM segment so that it cannot interact with the translocon. An implication of the deletion analysis of AcrB 265-AP is that amino acids 188 to 263 play a pivotal role in determining the targeting requirements of the protein (Fig. 2 and Fig. 4C and G). These data suggest that, in the absence of SRP, a large polypeptide segment beyond the TM domain is synthesized in the cytoplasm and can ultimately contribute to nonproductive folding. E. coli SRP appears to bind to the targeting sequences of nascent chains as soon as they are exposed to the cytoplasm (9), however, and probably prevents AcrB 265-AP from following a nonproductive pathway by targeting it to the membrane before a critical length of polypeptide is synthesized. IMPs and exported proteins may differ in their targeting requirements in part because the hydrophilic domains of IMPs have evolved to fold as membrane-anchored units and misfold if they interact with TM segments in the cytoplasm. Unlike IMPs, exported proteins appear to be well adapted to using SRP-independent targeting pathways. The available evidence suggests that signal sequences prevent the formation of a tightly folded structure and thereby maintain translocation competence after a large portion or all of a preprotein is synthesized (35).

Although our results indicate that the presence of a large hydrophilic domain plays an important role in determining the targeting requirements of an IMP, it is likely that the presence of multiple TM domains also contributes to the SRP dependence of polytopic IMP biogenesis. Unlike AcrB, some polytopic IMPs that require SRP for efficient insertion (e.g., lactate permease and -ketoglutarate permease) contain only relatively short hydrophilic tails and loops (48). Presumably SRP is required to target nascent polytopic IMPs to the membrane soon after synthesis of the first TM domain in order to prevent aggregation caused by the interaction of TM domains in the cytoplasm. Despite the prospect of misfolding or aggregation, however, the biogenesis of both bitopic and polytopic IMPs appears to be only partially SRP dependent. Even after Ffh was exhaustively depleted, the insertion of an IMP that contains both the P1 domain and seven TM domains (AcrB 576-AP) was reduced by only about 50%. Given that the concentration of Ffh has been estimated to be in the range of 200 to 1,000 copies per cell (21), it is likely that only a trace of Ffh remained after the 99.5% depletion that was achieved in our experiments. By contrast, a less-effective depletion of SecA in a closely related strain causes a quantitative block of AcrB 265-AP and AcrB 576-AP insertion, as well as protein export (39), indicating that much stronger effects are possible under certain experimental conditions. Partial SRP dependence might be due to a competition between the acquisition of an insertion-incompetent state, which is determined by the folding of a protein during its biosynthesis, and proper targeting of the protein by less-efficient mechanisms.

Considered together with the results of previous studies, the data presented here suggest an explanation for the essentiality of the SRP targeting pathway in E. coli (2, 27, 36). Although there appears to be a basal level of IMP insertion in the absence of SRP, the Ffh/4.5S RNA particle may play a critical role in cell physiology by simply expediting the delivery of nascent chains to the translocon. That is, cells may require SRP either because their growth is sensitive to the concentration of key IMPs that reside in the inner membrane or because the aggregation of IMPs in the cytoplasm that would result from the loss of SRP is highly toxic. Given that the chaperone-based targeting pathways may have evolved in rapidly growing organisms to increase the efficiency of protein translocation by facilitating the uncoupling of translation and translocation (22), it might have been expected that E. coli metabolism would be tuned to bypass the SRP pathway. Presumably, molecular chaperones cannot effectively duplicate the function of SRP although they, too, are thought to bind to exposed hydrophobic regions of passenger proteins (3). Indeed, the conservation of a specialized ribonucleoprotein-based targeting machinery suggests that chaperones cannot bind productively to IMPs or cannot deliver them to the translocon.