Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.     

Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.     

SecY variants that interfere with Escherichia coli protein export in the presence of normal secY

As an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY-d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose-binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild-type cells. The mutation deletes three amino acids that should be located at the interface of cytoplasmic domain 5 and transmembrane segment 9. We also found that some SecY-PhoA fusion proteins that lacked carboxy-terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy-terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the 'dominant negative' effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane.     

A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in Escherichia coli

Basic amino acid residues were introduced into an extracellular (periplasmic) domain, preceding a membrane-spanning hydrophobic domain, of SecY, an integral cytoplasmic membrane protein. The localization of the domain was monitored as to the alkaline phosphatase activity of TnPhoA fused adjacent to the domain. The alkaline phosphatase activity of such Escherichia coli cells drastically decreased when positive charges were introduced, indicating that on the introduction the SecY domain showed a change in localization from the periplasm to the cytoplasm. In another experiment, positive charges were introduced to the same periplasmic domain of another SecY-PhoA fusion protein, in which PhoA is fused to the cytoplasmic domain of SecY following the particular hydrophobic domain. The alkaline phosphatase activity increased drastically when positive charges were introduced, indicating that the SecY domain fused to PhoA showed a change in localization from the cytoplasm to the periplasm. In both experiments, the removal of a large amino-terminal portion of the SecY domain did not alter the effect of the positive charge introduction. Changes in localization of SecY domains thus demonstrated were also supported by a protease accessibility test on spheroplasts. It is proposed that a positively charged region adjacent to a membrane-embedded hydrophobic region tends to be stabilized on the cytoplasmic surface of the membrane, which in turn endows the hydrophobic region with the ability to act as a stop-transfer sequence or a signal sequence and consequently determines the orientation of the hydrophobic region in the membrane.     

Dislocation of membrane proteins in FtsH-mediated proteolysis.

Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.     

Deletion of the amino-terminal domain of asialoglycoprotein receptor H1 allows cleavage of the internal signal sequence

Human asialoglycoprotein receptor H1 is a single-spanning membrane protein with an amino-terminal domain of 40 residues exposed to the cytoplasm and the carboxyl-terminal domain translocated to the exoplasmic side of the membrane. It has been shown earlier that the transmembrane segment functions as an internal uncleaved signal sequence for insertion into the endoplasmic reticulum. In a deletion protein lacking almost the entire cytoplasmic domain, the signal sequence is cleaved at the carboxyl-terminal end of the transmembrane segment. All available criteria suggest that the protein is processed by signal peptidase. The cytoplasmic domain of the receptor does not directly inhibit signal cleavage since it does not detectably hinder cleavage of the normally amino-terminal signal sequence of influenza hemagglutinin in fusion proteins. We suggest that by its size or structure it affects the position of the receptor in the membrane and thus the accessibility of the potential cleavage site to signal peptidase.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation

The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for R357 in membrane protein insertion. Although R357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that R357 is essential for the initiation of SecA-dependent translocation only.     

Lack of effect of leader peptidase overproduction on the processing in vivo of exported proteins in Escherichia coli

The kinetics of maturation of certain exported proteins were analysed in Escherichia coli strains that also concomitantly overproduce either a periplasmic protein or the leader peptidase. The results led to three conclusions. Overproduction of leader peptidase has no effect on the rate of maturation of at least two exported proteins, one periplasmic (TEM beta-lactamase), one outer membrane (PhoE); therefore, the quantity of leader peptidase is not rate-limiting for normal export. Overproduction of PhoS reduces the rate of maturation of two other periplasmic proteins (beta-lactamase and PhoA) and itself, presumably by competing for the rate-limiting component of the export apparatus. Overproduction of leader peptidase in a strain overproducing PhoS has no effect on the retarded maturation of PhoS. Therefore even in these conditions, leader peptidase is not rate limiting.     

The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.     

The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

The role of the membrane electrochemical potential in the translocation of acidic and basic residues across the membrane was investigated with the M13 procoat protein, which has a short periplasmic loop, and leader peptidase, which has an extended periplasmically located N-terminal tail. For both proteins we find that the membrane potential promotes membrane transfer only when negatively charged residues are present within the translocated domain. When these residues are substituted by uncharged amino acids, the proteins insert into the membrane independently of the potential. In contrast, when a positively charged residue is present within the N-terminal tail of leader peptidase, the potential impedes translocation of the tail domain. However, an impediment was not observed in the case of the procoat protein, where positively charged residues in the central loop are translocated even in the presence of the membrane potential. Intriguingly, several of the negatively charged procoat proteins required the SecA and SecY proteins for optimal translocation. The studies reported here provide insights into the role of the potential in membrane protein assembly and suggest that electrophoresis can play an important role in controlling membrane topology.     

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.     

The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface.

The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins. VirB7 was shown to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with [3H]palmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II. A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a delta virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function. VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a delta virB operon derivative transformed with a virB7 expression plasmid. Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E. coli and A. tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts. VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A. tumefaciens spheroplasts and remained intact upon treatment of whole cells. Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space. Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell.     

Leader peptidase

The Escherichia coli leader peptidase has been vital for unravelling problems in membrane assembly and protein export. The role of this essential peptidase is to remove amino-terminal leader peptides from exported proteins after they have crossed the plasma membrane. Strikingly, almost all periplasmic proteins, many outer membrane proteins, and a few inner membrane proteins are made with cleavable leader peptides that are removed by this peptidase. This enzyme of 323 amino acid residues spans the membrane twice, with its large carboxyl-terminal domain protruding into the periplasm. Recent discoveries show that its membrane orientation is controlled by positively charged residues that border (on the cytosolic side) the transmembrane segments. Cleavable pre-proteins must have small residues at -1 and a small or aliphatic residue at -3 (with respect to the cleavage site). Leader peptidase does not require a histidine or cysteine amino acid for catalysis. Interestingly, serine 90 and aspartic acid 153 are essential for catalysis and are also conserved in a mitochondrial leader peptidase, which is 30.7% homologous with the bacterial enzyme over a 101-residue stretch.     

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.     

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.     

PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane

The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.     

Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)