External pH and nitrogen source affect secretion of pectate lyase by Colletotrichum gloeosporioides.

Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichum gloeosporioides: As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate. At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased. Nitrogen assimilation also was required for PL secretion at pH 6.0. Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0. Sequence analysis of the 5' upstream region of the pelB promoter revealed nine putative consensus binding sites for the Aspergillus transcription factor PacC. Consistent with this result, the transcript levels of pac1 (the C. gloeosporioides pacC homologue) and pelB increased in parallel as a function of pH. Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of C. gloeosporioides.

Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini.

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Alternative models for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24) has been substituted, yet the protein is processed efficiently and retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon. Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction.

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.

Expression and secretion of proteins in E. coli.

This review outlines approaches to the cloning and expression of proteins in Escherichia coli. The expression vectors described here (pIN-III derivatives) utilize the strong lipoprotein promoter, which is controlled by the lac-UV5 promoter-operator. These vectors provide the means for targeting a protein to any of the four subcellular compartments of the bacterial cell: cytoplasm, cytoplasmic membrane, periplasm, and outer membrane. Of particular importance is that secretion of proteins into the E. coli periplasm (using the OmpA signal peptide) is applicable for the production of both prokaryotic and eukaryotic proteins thereby enhancing protein activity and stability.

Import into mitochondria, folding and retrograde movement of fumarase in yeast.

A single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae. All fumarase translation products are targeted and processed in mitochondria before distribution. Here we show that targeting of fumarase is coupled to translation and initially involves insertion of the protein across the mitochondrial membranes and processing by the matrix protease. Rapid folding of fumarase may determine its requirement for coupling of its translocation with translation and unique route of distribution. The amino termini of most fumarase molecules are translocated across the mitochondrial membranes and processed. Unlike the in vivo situation where these molecules are released into the cytosol, in vitro they remain externally attached to the mitochondria, thereby positioned for release from the organelle. Our model suggests that fumarase displays a unique mechanism of targeting and distribution, which occurs cotranslationally and involves folding and retrograde movement of the processed protein back through the translocation pore.

Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae.

Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L-malic acid accumulation in the production medium. The high apparent Km of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function in the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production. The overexpression of MDH2 activity also causes an evaluation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.

The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase.

Saccharomyces cerevisiae accumulates L-malic acid but not only minute amounts of fumaric acid. A 13C-nuclear magnetic resonance study following the label from glucose to L-malic acid indicates that the L-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to L-malic acid is responsible for the L-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to L-malic but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards L-malic acid; the Km for L-malic acid is very high indicating that L-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more L-malic acid). (c) According to 13C NMR studies there is no interconversion of cytosolic L-malic and fumaric acids.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli. The N-terminal half of the yeast ABC protein Ste6.

Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli. To evaluate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast. Ste6, is an ATP binding cassette (ABC) protein, essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae. The topogenic reporters, invertase in S. cerevisiae and alkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. The results obtained in both systems are similar, although more definitive in E. coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6. Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar. In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.

The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUM1), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUM1 messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUM1 gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol.

Characterization of the rnc-97 mutation of RNAaseIII: a glycine to glutamate substitution increases the requirement for magnesium ions.

The rnc-97 mutation of the Escherichia coli double-stranded-RNA-specific ribonuclease III (RNAaseIII) was previously isolated by virtue of the lethal expression of RNAaseIII in Saccharomyces cerevisiae. Here we show that rnc-97 is a single point mutation causing the substitution of glycine 97 by glutamic acid. The mutation eliminates the lethal phenotype of RNAaseIII expression in yeast and reduces fourfold the effect of RNAaseIII expression on bacteriophage gy1 propagation in E. coli. Mutant RNAaseIII-G97E and wild-type RNAaseIII were purified according to published procedures. The apparent molecular masses of the two enzymes on SDS polyacrylamide gels are the same but they differ in pI (6.85 for RNAaseIII-G97E and 7.3 for RNAaseIII). Whereas the two enzymes (under standard assay conditions) do not show a great difference in activity towards double-stranded RNA and defined single-stranded RNAaseIII substrates, they differ dramatically (20-fold or more) under conditions of Mg2+ limitation. The hypothesis that limitation of Mg2+ ions in vivo is responsible for the phenotypes of the rnc-97 mutation in S. cerevisiae and E. coli is discussed.

The relationship between disulphide bond formation, processing and secretion of lipo-beta-lactamase in yeast.

The hybrid prokaryotic lipo-beta-lactamase mature and precursor proteins spontaneously form an intramolecular disulphide bond when oxidized in vitro. When expressed in Saccharomyces cerevisiae (in vivo) the lipo-beta-lactamase precursor is in a reduced form whereas the majority of the mature protein is oxidized. The results indicate that in yeast, the lipo-beta-lactamase precursor is first processed (the signal peptide is removed) and then oxidized to form a disulphide bond in the mature protein. Reduced-mature lipo-beta-lactamase was found to reach the yeast periplasm and the process depends on endoplasmic reticulum (ER) entry even though the protein is not oxidized. This result is remarkable since in eukaryotes, disulphide bond formation occurs in the ER. Oxidized mature lipo-beta-lactamase can also be released from the sphaeroplast into the yeast periplasm. Mutant lipo-beta-lactamase genes in which cysteine residue 131 was changed to either tyrosine or threonine, were efficiently processed and secreted in yeast, which is consistent with the finding that reduced-mature non-mutant lipo-beta-lactamase can be secreted. We discuss the possibility that the folding mechanism of lipo-beta-lactamase in vitro may be fundamentally different from the process in the eukaryotic system of S. cerevisiae.

Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae.

A hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat-labile enterotoxin (Lipo-EtxB) was expressed in yeast and E. coli. Analyses of cell lysates from Saccharomyces cerevisiae and E. coli expressing the protein revealed that both organisms were able to assemble Lipo-EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1-ganglioside receptors. Each of these properties are characteristic of the wild-type B subunit pentamer produced in E. coli. Assembly of Lipo-EtxB was found to be unaffected in a sec18 mutant of S. cerevisiae, which possesses a temperature-sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER. A kar2-1 mutation with a defect in the yeast homologue of BiP caused an 18-fold reduction in Lipo-EtxB assembly at the non-permissive temperature in S. cerevisiae. However, introduction of the wild-type KAR2 gene on a plasmid into the kar2-1 mutant completely suppressed the inhibition of Lipo-EtxB assembly. This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a 'molecular chaperone'. The possible mechanisms of enterotoxoid assembly in E. coli and S. cerevisiae are discussed.

Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences.

Staphylococcus aureus nuclease A hybrid genes, encoding proteins OmpA-nuclease, lipo-nuclease and Pin-nuclease, were cloned downstream of the yeast GAL10 inducible promoter. OmpA-nuclease and lipo-nuclease contain the mature staphylococcal nuclease sequence preceded by the Escherichia coli OmpA and lipoprotein signal sequences, respectively, whereas Pin-nuclease lacks a defined signal sequence at its amino terminus. We found that: (a) the nuclease gene products synthesized in yeast are active, but they do not affect cell growth; (b) OmpA-nuclease and lipo-nuclease are partially processed and constitute approximately 1.0-1.5% of the yeast cell protein; (c) OmpA and lipoprotein signal sequences function similarly in secretion, allowing 35-40% of the processed nuclease to be translocated into the yeast periplasm; and (d) Pin-nuclease, which lacks hydrophobic sequences at its amino-terminus, is accumulated at a level tenfold lower than the hybrid proteins that do contain signal sequences. Nevertheless, 50% of the enzyme activity of Pin-nuclease in yeast is localized in the periplasmic space.

Specific beta-lactam antibiotics inhibit secretion of lipo-beta-lactamase in yeast.

The beta-lactam antibiotic cloxacillin can inhibit secretion of prokaryotic lipo-beta-lactamase into the periplasm of yeast. The results indicate that this phenomenon is specific with respect to both the antibiotic and the lipo-beta-lactamase whose secretion is affected, strongly suggesting that this involves an interaction between the enzyme and its substrates. The effect of the antibiotic on secretion is reversible. With different beta-lactam antibiotics, the clearest difference is observed between type A and type S penicillins; the former exert a strong inhibition of secretion whereas the latter exhibit a weak effect or no effect at all. Type A penicillins have been previously shown to cause a conformational change in various beta-lactamases. Mature lipo-beta-lactamase species in yeast were localized either to the periplasmic space or bound to the outer surface of the cytoplasmic membrane and thus exposed to periplasm. The results are consistent with the hypothesis that binding of cloxacillin to lipo-beta-lactamase induces a conformation on the protein that is unfavourable for its release from the membrane.

Inducible overexpression of the FUM1 gene in Saccharomyces cerevisiae: localization of fumarase and efficient fumaric acid bioconversion to L-malic acid.

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.

Expression of double-stranded-RNA-specific RNase III of Escherichia coli is lethal to Saccharomyces cerevisiae.

The gene for the double-stranded RNA (dsRNA)-specific RNase III of Escherichia coli was expressed in Saccharomyces cerevisiae to examine the effects of this RNase activity on the yeast. Induction of the RNase III gene was found to cause abnormal cell morphology and cell death. Whereas double-stranded killer RNA is degraded by RNase III in vitro, killer RNA, rRNA, and some mRNAs were found to be stable in vivo after induction of RNase III. Variants selected for resistance to RNase III induction were isolated at a frequency of 4 X 10(-5) to 5 X 10(-5). Ten percent of these resistant strains had concomitantly lost the capacity to produce killer toxin and M dsRNA while retaining L dsRNA. The genetic alteration leading to RNase resistance was localized within the RNase III-coding region but not in the yeast chromosome. These results indicate that S. cerevisiae contains some essential RNA which is susceptible to E. coli RNase III.

Defective Escherichia coli signal peptides function in yeast.

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.

The isolation and characterization of RNA coded by the micF gene in Escherichia coli.

A new species of micF RNA, which contains 93 nucleotides (a 4.5S size), was isolated from Escherichia coli. The sequence of the 4.5S micF RNA corresponds to positions G82 through U174 of the micF gene. The 5' terminal end of this smaller micF RNA is triphosphorylated signifying that it is a primary transcript. Its promoter region, which is situated within the greater micF structural gene, has been identified and characterized by lacZ fusion analysis. A 6S micF RNA species, which has a base composition predicted for a transcript from the full length gene has also been detected; however, the 4.5S micF RNA is the predominant species. The work clearly shows by biochemical identification the presence of chromosomally encoded micF RNA.