Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein.

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles.     

Import of honeybee prepromelittin into the endoplasmic reticulum: energy requirements for membrane insertion.

The import of small precursor proteins, derived from the honeybee secretory protein prepromelittin, into dog pancreas microsomes is independent of signal recognition particle (SRP) and docking protein, but requires that charged amino acids at the amino terminus of the mature part are counterbalanced by amino acids with the opposite charge at the carboxy terminus. The import pathway of such precursor proteins was resolved into two sequential steps: (i) binding of precursors to microsomes, and (ii) insertion of precursors into the membrane. Formation of an intramolecular disulfide bridge within the mature part of these precursor proteins allowed association of the oxidized precursors with the microsomal membrane but reversibly inhibited their membrane insertion. Furthermore, membrane insertion was inhibited by ATP depletion. Different prepromelittin derivatives were found to depend on ATP to varying degrees. We conclude that insertion of prepromelittin-derived precursor proteins into microsomal membranes involves a competent conformation of the precursor proteins and that, in general, this is accomplished with the help of both a cytoplasmic component and ATP.     

Structural requirements for transport of preprocecropinA and related presecretory proteins into mammalian microsomes.

The presecretory protein preprocecropinA (which comprises 64 amino acid residues) as well as a synthetic hybrid between preprocecropinA and dihydrofolate reductase (which comprises 252 amino acid residues) are processed by and transported into mammalian microsomes. Transport of both precursor proteins can take place cotranslationally, i.e. with the aid of ribosome and signal recognition particle, or posttranslationally, i.e. independently of these ribonucleoparticles (RNPs). We investigated the role of the precursor structure with respect to competence for RNP-independent transport by constructing deletion mutants and hybrid proteins. The results demonstrate that the signal peptide is essential for RNP-independent transport. Furthermore, the signal peptide is sufficient for translocation of preprocecropinA derivatives up to 85 amino acid residues in size. However, the conformation of the precursor protein is decisive in the case of larger hybrid proteins.     

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.     

Ribonucleoparticle-independent transport of proteins into mammalian microsomes

There are at least two different mechanisms for the transport of secretory proteins into the mammalian endoplasmic reticulum. Both mechanisms depend on the presence of a signal peptide on the respective precursor protein and involve a signal peptide receptor on the cis-side and signal peptidase on the trans-side of the membrane. Furthermore, both mechanisms involve a membrane component with a cytoplasmically exposed sulfhydryl. The decisive feature of the precursor protein with respect to which of the two mechanisms is used is the chain length of the polypeptide. The critical size seems to be around 70 amino acid residues (including the signal peptide). The one mechanism is used by precursor proteins larger than about 70 amino acid residues and involves two cytosolic ribonucleoparticles and their receptors on the microsomal surface. The other one is used by small precursor proteins and relies on the mature part within the precursor molecule and a cytosolic ATPase.     

Expression of honeybee prepromelittin as a fusion protein in Escherichia coli.

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.     

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.     

Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of approximately 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.     

Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli

Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.     

DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum.

The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli.     

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane.

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.     

The signal sequence of an Escherichia coli outer membrane protein can mediate translocation of a not normally secreted protein across the plasma membrane

The distal part of the long tail fibers of the Escherichia coli phage T4 consists of a dimer of protein 37. A fragment of the corresponding gene, encoding 253 amino acids, was inserted into several different sites within the cloned gene for the 325-residue outer membrane protein OmpA. In plasmid pTU T4-5 the fragment was inserted once and in pTU T4-10 tandemly twice between the codons for residues 153 and 154 of the OmpA protein. In pTU T4-22 two fragments were present, in tandem, between the codons for residues 45 and 46 of this protein. In pIN T4-6 one fragment was inserted into the ompA gene immediately following the part encoding the signal sequence. The corresponding mature proteins consist, in this order, of 605, 860, 835, and 279 amino acid residues. All precursor proteins were processed and translocated across the plasma membrane. Hence, not only can the OmpA protein serve as a vehicle for export of a nonsecretory protein, but the signal sequence alone can also mediate export of such a protein. Export of the pro-OmpA protein depends on the SecA protein. Export of the tail fiber fragment expressed from pIN T4-6 remained SecA dependent. Thus, the secA pathway in this case is chosen by the signal peptide. It is proposed that a signal peptide can mediate translocation of nonsecretory proteins as long as they are export-compatible. The inability of a signal sequence to mediate export of some proteins appears to be due to export incompatibility of the protein rather than to the absence of information, within the mature part of the polypeptide, which would be required for translocation.     

Signal peptide mutants ofEscherichia coli

Numerous secretory proteins of the Gram-negative bacteriaE. coli are synthesized as precursor proteins which require an amino terminal extension known as the signal peptide for translocation across the cytoplasmic membrane. Following translocation, the signal peptide is proteolytically cleaved from the precursor to produce the mature exported protein. Signal peptides do not exhibit sequence homology, but invariably share common structural features: (1) The basic amino acid residues positioned at the amino terminus of the signal peptide are probably involved in precursor protein binding to the cytoplasmic membrane surface. (2) A stretch of 10 to 15 nonpolar amino acid residues form a hydrophobic core in the signal peptide which can insert into the lipid bilayer. (3) Small residues capable of -turn formation are located at the cleavage site in the carboxyl terminus of the signal peptide. (4) Charge characteristics of the amino terminal region of the mature protein can also influence precursor protein export. A variety of mutations in each of the structurally distinct regions of the signal peptide have been constructedvia site-directed mutagenesis or isolated through genetic selection. These mutants have shed considerable light on the structure and function of the signal peptide and are reviewed here.     

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.     

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.     

Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N-terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N-terminus of Oxa1p (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N-terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial-type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.     

Import of honeybee prepromelittin into the endoplasmic reticulum. Requirements for membrane insertion, processing, and sequestration

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Membrane insertion of prepromelittin, assayed as signal sequence removal by signal peptidase, is not dependent on signal recognition particle and docking protein. However, a previously uncharacterized proteinaceous component of the microsomal membrane is required for completion of membrane transfer of promelittin. Furthermore, membrane insertion of prepromelittin is not coupled to translation. These data suggest the signal sequence, in addition to its role in membrane recognition, has a more general function for membrane insertion, cotranslational import of proteins is not an intrinsic feature of microsomes, and at least in certain cases, proteinaceous membrane components are involved in membrane transfer.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

Regulation and structure of an Escherichia coli gene coding for an outer membrane protein involved in export of K88ab fimbrial subunits.

The nucleotide sequence of the faeD gene of Escherichia coli and the amino acid sequence of its product is presented. The faeD product is an outer membrane protein required for transport of K88ab fimbrial subunits across the outer membrane. The protein is synthesized as a precursor containing a signal peptide, and the tentative mature protein comprises 777 amino acid residues. The distribution of amino acids in the faeD protein is similar to that of other outer membrane proteins; showing a fairly even distribution of charged residues and the absence of extensive hydrophobic stretches. Secondary structure predictions revealed a region of 250 amino acid residues which might be embedded in the outer membrane. The 5'-end of faeD is located within a region showing dyad symmetry. This region serves to couple translation of faeD to the translation of the gene preceding it (faeC). The 3'-end of faeD shows an overlap of 5 bases with the next gene (faeE).     

Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.

The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.