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.
Schonberger
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Department of Molecular Biology, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Hirst
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Pines
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D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004768:Enterotoxins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D008074:Lipoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C027538:heat-labile enterotoxin, E coli; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; C072713:SEC53 protein, S cerevisiae