The secY protein can act post-translationally to promote bacterial protein export

Conditionally lethal Escherichia coli mutants in secY (prlA) show defective export of proteins to the periplasm and outer membrane. It has been proposed that this gene and other sec genes must act on pro-OmpA at an early stage of protein synthesis in order to allow later translocation to occur. We have described a temperature-sensitive mutation in which the secYts function is impaired at the nonpermissive temperature (Ito, K. (1984) Mol. Gen. Genet. 197, 204-208). A plasmid bearing the wild-type secY gene under the control of the lactose operon (Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635) has been introduced into this mutant strain. We now report that the in vivo chase of pulse-labeled full length pro-OmpA to mature OmpA is accelerated by inducing the synthesis of the wild-type secY protein at the end of the period of pulse labeling. We have also assayed the requirements for secY function for in vitro protein translocation. Membranes derived from secY ts cells which were incubated at 42 degrees C were inactive in vitro in the post-translational uptake and processing of pro-OmpA. Thus, the secY protein can act post-translationally, enhancing the translocation of completed pro-OmpA polypeptide chains across the plasma membrane.     

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

Overproduction, isolation and determination of the amino-terminal sequence of the SecY protein, a membrane protein involved in protein export in Escherichia coli

The gene product of secY (prlA) is an integral membrane protein with an essential role in protein export in Escherichia coli. When the protein was overproduced, using a plasmid, it was degraded rapidly in the cell. The lon or the htpR mutation did not slow down this degradation, but low-temperature growth conditions (30 degrees C) did so appreciably. On the other hand, the copy number of the pUC8-based plasmid was higher at higher temperatures. Thus, the plasmid was first amplified at 42 degrees C and the protein was then accumulated at 30 degrees C. The SecY protein was isolated in sodium dodecyl sulfate (SDS)-denatured form from the membranes of the overproducing cells, using SDS-SDS two-dimensional gel electrophoresis. Its NH2-terminal sequence confirmed the secY reading frame and the translation initiation site assigned previously. The SecY protein does not undergo NH2-terminal processing except for the removal of the initiator methionine.     

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.     

Accumulation of prolipoprotein in Escherichia coli mutants defective in protein secretion.

The export of lipoprotein has been found to be affected in both secA and secY mutants of Escherichia coli which are defective in the secretion of a number of outer membrane and periplasmic proteins. The kinetics of accumulation of prolipoprotein upon a temperature shift to 42 degrees C is indistinguishable from that of pre-OmpA protein accumulation in the secA mutant. In both secA and secY mutants, the accumulated prolipoprotein is unmodified with glyceride and localized in the cytoplasmic membrane. We conclude from these results that the early steps in protein export are common to prolipoprotein and non-lipoprotein precursors. The pathways for the export of these two groups of precursor proteins diverge with regard to the modification and processing reactions which are late events in the export process.     

Mutation that suppresses the protein export defect of the secY mutation and causes cold-sensitive growth of Escherichia coli.

A cold-sensitive mutant was isolated among temperature-resistant revertants of the secY24 mutant defective in secretion of envelope proteins across the cytoplasmic membrane at 42 degrees C. A single mutation, designated ssyA3, is responsible both for the extragenic suppression of secY and for the cold-sensitive growth. In contrast to the parental secY24 mutant, the suppressed cells do not accumulate precursors of envelope proteins at any temperatures. The cells containing the ssyA3 mutation, whether in combination with secY24 or not, show an optimal growth at 42 degrees C and a very poor growth at 30 degrees C. At the low temperature, protein synthesis is generally slowed down, probably at the step of chain elongation. The gene ssyA was mapped at a new locus between hisS and glyA on the chromosome. It is possible that the product of this gene interacts both with the protein secretion system and the protein synthesizing system.     

Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coli.

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564-5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.     

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.     

Novel prlA alleles defective in supporting staphylokinase processing in Escherichia coli

A class of prlA (secY) alleles of Escherichia coli (prlA4-1 and prlA401) which specifically block the export of staphylokinase has been identified (T. Iino and T. Sako, J. Biol. Chem. 263:19077-19082, 1988; T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). To determine more precisely the region in PrlA (SecY) effective for the blockage of processing of the staphylokinase precursor, additional prlA mutants which failed to support processing of the staphylokinase precursor were isolated. Two of the five mutant alleles isolated (secY121 and secY161) complemented the temperature sensitivity of a secY24 strain and had no detectable effect on the processing of endogenous secretory proteins of E. coli. In addition, a staphylokinase mutant having glycine in place of serine at position 17 in its signal sequence relieved the detrimental effect of these mutations. All of these characteristics indicate that these two alleles resemble the prlA4-1 and prlA401 alleles. On the other hand, the remaining three mutant alleles (secY47, secY105, and secY112) had no significant PrlA activity. The mutations of secY121 and secY161 were mapped very close to those of prlA4-1 and prlA401 in the presumed transmembrane segment 7 of PrlA. These results indicate that transmembrane segment 7 of PrlA plays a crucial role in the recognition of the staphylokinase signal sequence.     

The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins.

The product of the secY (prlA) gene (the SecY protein) involved in protein export in Escherichia coli was overproduced and localized in the cytoplasmic (inner) membrane. Because of its strong interaction with a non-ionic detergent (NP40), it partitioned into the detergent layer during electroblotting through a NP40-containing gel (detergent blotting), and it formed a horizontal streak in the O'Farrell two-dimensional gel electrophoretic system. Consequently, we developed an alternative two-dimensional gel procedure, which proved useful for analysis of integral membrane proteins, especially in combination with detergent blotting. SDS-gel electrophoresis was carried out successively through gels of lower (first dimension) and higher (second dimension) sieving effects. Many membrane proteins, unlike soluble proteins, formed spots off and above the diagonal line, and all of these spots partitioned exclusively into the detergent layer. A characteristic pattern of integral membrane proteins of E. coli was thus obtained and the spot of the SecY protein in the cytoplasmic membrane was identified even when it was not overproduced. These results show that the gene secY specifies an integral membrane component of the protein export machinery.     

Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli

We have examined the effects of thermosensitive mutations in secA and secY (prlA) genes on the export of proteins to the three layers of the Escherichia coli cell surface. After several hours at the nonpermissive temperature, the export of two major outer membrane proteins, lipoprotein and OmpA, is delayed, then essentially blocked, in either a secA or secY strain. These mutations also have a strong effect on the export of several proteins, such as maltose binding protein, to the periplasm, though the export of many periplasmic proteins is not affected. secA and secY block the assembly of leader peptidase, which is made without a leader sequence, into the inner membrane. However, the membrane assembly of M13 coat protein (an inner membrane protein made with an amino-terminal leader sequence) is not affected. Thus, the requirement for sec function for export does not correlate with the presence or absence of leader peptide or with a particular subcellular compartment, but rather is specific to each particular protein.     

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli

We have previously described a temperature-sensitive mutant, ts215, which is defective in protein secretion. Complementation studies indicated that the mutation was located at the distal part of the spc ribosomal protein operon and the gene secY is required for efficient protein secretion. We now report a more complete genetic and biochemical analysis of the ts215 mutant. These studies revealed that the ts215 mutant has an amber mutation in the gene rp10 for ribosomal protein L15, which is located upstream and adjacent to secY. The amber mutation exerts a polar effect on secY causing a defect in protein secretion. These conclusions were supported by the following observations. The mutant strain carries a phi 80 prophage containing a temperature-sensitive suppressor, supFts6. The strain contains decreased amounts of L15 and is suppressible by a temperature-independent nonsense suppressor. In addition, L15 contains an extra tyrosine residue when suppressed by supF. DNA sequence analysis revealed the presence of a single base change in rp10 resulting in an amber codon at the 38th codon of L15. The mutant phenotype is complemented by a plasmid carrying only the secY gene under lac promoter control. The mutant cells complemented by secY can grow and synthesize proteins at normal rates and abundances at 42 degrees C, despite the fact that their ribosomes contain barely detectable levels of L15. These results indicate that ribosomal protein L15 is dispensable for protein synthesis and cell growth. In contrast, the decreased level of expression of the secY gene leads to defective protein secretion and defective cell growth.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.     

Distinct mutation sites in prlA suppressor mutant strains of Escherichia coli respond either to suppression of signal peptide mutations or to blockage of staphylokinase processing.

We have cloned and sequenced some prlA mutant alleles of the Escherichia coli secY gene. From the mutation sites determined, it is strongly suggested that distinct regions in the SecY (PrlA) protein are involved in the recognition of different structural features of a signal peptide as it functions.     

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.     

Disruption of rpmJ encoding ribosomal protein L36 decreases the expression of secY upstream of the spc operon and inhibits protein translocation in Escherichia coli

The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a rho-independent terminator is inserted immediately downstream of secY. Moreover, it has been suggested that RpmJ is dispensable for E. coli. We constructed rpmJ null strains, AY101 (DeltarpmJ::tetA) and AY201 (DeltarpmJ::cat), by replacing rpmJ with tetA, which encodes a membrane protein responsible for tetracycline-resistance, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Depletion of RpmJ did not inhibit protein synthesis, whereas the growth of AY101 was defective at high temperatures. The level of SecY mRNA decreased significantly in both disruptants even though the rho-independent terminator was inserted immediately downstream of secY. Some periplasmic proteins were missing in the disruptants with a concomitant increase in the amount of phage shock protein in the inner membrane. These phenotypes caused by the rpmJ null mutation were corrected by a plasmid carrying secY, but not by one carrying rpmJ.     

Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli.

To define additional components of the export machinery of Escherichia coli, I have isolated extragenic suppressors of a mutant [secA(Ts)] that is temperature sensitive for growth and secretion at 37 degrees C. Suppressors that restored growth at 37 degrees C, but that rendered the cell cold sensitive for growth at 28 degrees C, were obtained. The suppressor mutations fall into at least seven loci, two of which (prlA and secC) have been previously implicated in protein secretion. The five remaining loci (ssaD, ssaE, ssaF, ssaG, and ssaH) have been mapped by P1 transduction and appear to define new genes in E. coli. All of the suppressor mutations allow both enhanced growth and protein secretion of the secA(Ts) mutant at 37 degrees C, but not 42 degrees C, indicating a continued requirement for SecA protein. Strains carrying solely the cold-sensitive mutations show reduced levels of certain periplasmic proteins when grown at low temperatures. In at least one case, that of maltose-binding protein, this defect is at the level of synthesis of the protein. Since mutants in any of seven genes as well as secA amber mutants halt or reduce the synthesis of an exported protein, it appears that E. coli may possess a general and complex mechanism for coupling protein synthesis and secretion.     

Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli

To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.     

Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors.

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.