Protein Export Pathway Research Articles

A new pathway for protein export in Saccharomyces cerevisiae.

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

FliH and FliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria

Two motility genes ( fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced. FliH and FliI showed extensive homology to the proteins involved in the export of glagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

A nuclear export signal in hnRNP A1: A signal-mediated, temperature-dependent nuclear protein export pathway

Pre-mRNAs are associated with hnRNPs, and these proteins play important roles in the biogenesis of mRNAs. The hnRNP A1 is one of the most abundant hnRNPs, and although localized primarily in the nucleoplasm, shuttles continuously between the nucleus and the cytoplasm. A 38 amino acid domain within A1, termed M9, which bears no resemblance to classical nuclear localization signal (NLS) sequences, localizes A1 to the nucleus. Here we show that M9 is also a nuclear export signal; placing M9 on a protein that is otherwise restricted to the nucleus, the nucleoplasmin core domain (NPc), efficiently exports it to the cytoplasm in a temperature-dependent manner. In contrast, classical NLSs cannot promote the export of NPc. These findings demonstrate that there is a signal-dependent, temperature-sensitive nuclear export pathway and strengthen the suggestion that A1 and other shuttling hnRNPs function as carriers for RNA during export to the cytoplasm.

Rapid purification of native SecA from Escherichia coli: development of a new affinity chromatography procedure.

The SecA protein occupies a pivotal position in the public protein export pathway in Escherichia coli. The multifunctional SecA protein recognizes cytoplasmic factors associated with export including the presecretory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of SecA to bind ATP was the basis for the development of a novel, rapid purification scheme involving a single chromatographic step. Affinity chromatography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts of E. coli binds strongly to this dye-ligand matrix, and active protein was purified to greater than 90% homogeneity. The protein isolated by this procedure retained the previously described ATPase and RNA-binding activities of SecA. This approach should permit the rapid purification of SecA homologs from a variety microorganisms.

Molecular evolution of SRP cycle components: functional implications.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane. We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP. Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway. The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution. The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria. Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein. Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif. Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E. coli EF-Tu. The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha. Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases. The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin.

Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens.

FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage. FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A. P. Volger, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). It is even more similar to the Spa47 protein of Shigella flexneri (M. M. Venkatesan, J. M. Buysse, and E. V. Oaks, J. Bacteriol. 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S. Fenselau, I. Balbo, and U. Bonas, Mol. Plant-Microbe Interact. 5:390-396, 1992), which are believed to play a role in the export of virulence proteins. Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase. FliI was overproduced and purified almost to homogeneity. It demonstrated ATP binding but not hydrolysis. An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell. An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related. The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.

The Cs sec mutants of Escherichia coli reflect the cold sensitivity of protein export itself.

We have found that temperature can have a striking effect upon protein export in Escherichia coli, suggesting that there is a cold-sensitive step in the protein export pathway. Cs mutations comprise the largest class of mutations affecting the membrane-localized Sec proteins SecD, SecE, SecF and SecY. Although some of these mutations could encode cold-labile proteins, this is unlikely to account for the Cs phenotype of most export mutants, as mutations which simply produce lower amounts of SecE protein have the same phenotype. Certain signal sequence mutations affecting maltose binding protein are also cold sensitive for export. These effects appear to arise by a specific interaction of cold with certain export defects. We believe that the Cs sec mutations are representative of a large class of conditional lethal mutations, whose conditional phenotype reflects an underlying thermal sensitivity of the process in which they are involved.

SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane.

Recent insight into the biochemical mechanisms of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to inner-membrane anionic phospholipids, insertion into the membrane bilayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein export pathway. This form of negative regulation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways.

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane.

Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria

Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces. Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage. We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region was physically mapped to a SpeI fragment around 20 min on the P. aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene ( fimA/ pilA) and ancillary genes required for fimbrial assembly ( pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K. pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid. We have also identified other sets of homologies between P. aeruginosa fimbrial assembly (Pil) proteins and B. subtilis Com and K. oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria.

Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells.

Saccharomyces cerevisiae MATa cells release a lipopeptide mating pheromone, a-factor. Radiolabeling and immunoprecipitation show that MATa ste6 mutants produce pro-a-factor and mature a-factor intracellularly, but little or no extracellular pheromone. Normal MATa cells carrying a multicopy plasmid containing both MFa1 (pro-a-factor structural gene) and the STE6 gene secrete a-factor at least five times faster than the same cells carrying only MFa1 in the same vector. The nucleotide sequence of the STE6 gene predicts a 1290 residue polypeptide with multiple membrane spanning segments and two hydrophilic domains, each strikingly homologous to a set of well-characterized prokaryotic permeases (including hlyB, oppD, hisP, malK and pstB) and sharing even greater identity with mammalian mdr (multiple drug resistance) transporters. These results suggest that the STE6 protein in yeast, and possibly mdr in animals, is a transmembrane translocator that exports polypeptides by a route independent of the classical secretory pathway.

Escherichia coli SecB protein associates with exported protein precursors in vivo.

The product of the Escherichia coli secB gene is required for efficient export of proteins across the cytoplasmic membrane. The studies described in this report show that in wild-type growing cells, SecB protein associates with precursor forms of exported proteins, such as the periplasmic maltose-binding protein (MBP) and the outer-membrane proteins LamB and OmpA. In contrast, the cytoplasmic protein beta-galactosidase was not found in association with SecB. Pulse-chase analysis showed that the SecB-precursor MBP complex was short lived, as expected for a complex that represents an intermediate in the protein-export pathway. The results support the hypothesis that SecB protein associates with exported protein precursors in the cytoplasm and dissociates prior to or during translocation of precursors across the cell membrane.

Klebsiella pneumoniae pulS gene encodes an outer membrane lipoprotein required for pullulanase secretion

The product of the Klebsiella pneumoniae gene pulS, which is located downstream from the pullulanase structural gene (pulA), is essential for the cell surface localization and extracellular release of pullulanase in Escherichia coli K-12. pulS is transcribed in the opposite direction to pulA, from which it is separated by a region of 624 nucleotides. Although this latter region contains a new component of the maltose regulon, pulB, which is transcribed from the pulA promoter, it is not required for pullulanase synthesis or secretion. Unlike pulA and all other pullulanase secretion genes characterized so far, the expression of pulS is not induced by growth in the presence of maltose and is unaffected by mutations in the maltose regulator gene malT. The pulS gene product was identified as a ca. 12-kilodalton outer membrane lipoprotein. The characterization of PulS brings to three the number of identified proteins which are known to be required for pullulanase secretion in addition to the components of the signal sequence-dependent general protein export pathway.

The secE gene encodes an integral membrane protein required for protein export in Escherichia coli.

Genetic screening and selection procedures employing a secA-lacZ fusion strain repeatedly have yielded mutations in four genes affecting the protein export pathway of Escherichia coli. These genes are secA, secD, prlA/secY, and secE. We discuss the significance of the failure to find new sec genes after extensive use of this approach. One of the genes, secE, has been characterized in some detail. From the DNA sequence of the gene and analysis of alkaline phosphatase fusions to the SecE protein, we propose that it is a 13,600-dalton integral cytoplasmic membrane protein. The data presented here and in the accompanying paper strongly suggest that secE has an important role in E. coli protein export.

The gene encoding the gIII envelope protein of pseudorabies virus vaccine strain Bartha contains a mutation affecting protein localization

Pseudorabies virus (PRV) vaccine strain Bartha has a diminished capacity to cause disease and harbors a variety of mutations affecting virulence. It has been reported that PRV Bartha produces virions with reduced amounts of the major envelope glycoprotein gIII. One hypothesis was that this phenotype was due to reduced expression of the gIII gene. In this report, we demonstrate that the reduced amount of gIII in virions was not mediated at the level of transcription, but rather reflected a defect in protein localization. We describe experiments with gene replacement technology to prove that the expression defect was closely linked to the gIII gene itself. Using pulse-chase experiments, we found a defect similar to that observed for certain signal sequence mutations of PRV Becker gIII. The Bartha gIII protein was translated, but was inefficiently introduced into the membrane protein export pathway. Consequently, only a fraction of the primary Bartha gIII translation product was glycosylated and matured. The remaining fraction stayed presumably in the cytoplasm, where it never became glycosylated or inserted into cell or virus membranes. The result was that Bartha-infected cells produced virions with reduced amounts of gIII in their envelopes. Comparison of the DNA sequence of the promoter and amino-terminal coding regions of Becker and Bartha gIII genes revealed a single base pair difference in Bartha, changing codon 14 of the signal sequence from a leucine (CTC) to a proline (CCC) codon. We suggest that the signal sequence mutation is responsible for the apparent reduced expression phenotype of this attenuated strain. This mutation represents, to our knowledge, the first reported natural signal sequence mutation in a herpesvirus glycoprotein.

Expression of human preproapo AI and pre(delta pro)apoAI in a murine pituitary cell line (AtT-20). A comparison of their intracellular compartmentalization and lipid affiliation.

The role of the NH2-terminal propeptide of human apolipoprotein (apo) AI in intracellular transport and lipid-protein interactions was examined by transfecting a murine anterior pituitary cell line (AtT-20) with the human preproapo AI gene and a mutant gene lacking the 18-base pair segment of exon III encoding its hexapeptide prosegment. ProapoAI was not processed to the mature apolipoprotein either prior to or after export from these cells making this an attractive model system for directly assessing structure/activity relationships of its propeptide. ApoAI was sorted into a regulated pathway for protein export. The signal responsible for this trafficking pattern was not contained in the prosegment since both pro- and mature ApoAI exhibited a similar rate of secretion, a similar magnitude of stimulation of export by the secretagogue 8-bromo-cyclic AMP, and similar targeting to dense core granules. This sorting behavior, exhibited by a protein which is not normally targeted to dense core secretory granules in its cells of origin, raises questions about the domains and mechanisms involved in protein sorting into the regulated and constitutive pathways of AtT-20 cells. Density gradient ultracentrifugation, immunoaffinity chromatographic and electron microscopic analysis indicated that approximately 10% of proapoAI and approximately 10% apoAI appeared in AtT-20 culture media in the form of two discrete classes of nascent lipoproteins: a small 6-8 nm spherical particle and a larger discoidal particle. These particles had morphologies identical with those secreted by Hep G2 cells which normally synthesize apolipoproteins. Although these results do not resolve the issue of whether or not a fraction of apoAI can act as an acceptor of cellular lipid during its transport through the secretory pathway, the data do show that this functional capability for lipoprotein assembly is not obviously regulated by its prosegment.

Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope

An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently. Consequently, such a strain is defective for the utilization of maltose as a sole carbon source. We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein. A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E. coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map. Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope. Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur. Our data suggest that proper assembly of a complex is required for efficient export of E. coli envelope proteins to their various extracytoplasmic compartments.