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 enterotoxrn (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‐ganglio‐side 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 KAR2gene 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.