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.
Shani
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O
Department of Molecular Biology, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Pines
O
O
D000595:Amino Acid Sequence; D001483:Base Sequence; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004220:Disulfides; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; C037640:pre-beta-lactamase; D001618:beta-Lactamases