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Cassette mutagenic analysis of the signal peptide of yeast invertase Ngsee, Johnny Kuan


The SUC2 locus of Saccharomyces cerevisiae encodes two forms of invertase; a constitutively expressed cytoplasmic enzyme and a glucose-repressible secreted and glycosylated enzyme which is initially produced with an amino-terminal signal peptide. The coding sequence of the SUC2 locus has been placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere based yeast plasmid vector from which invertase is expressed in a Sue" strain of yeast. Oligonucleotide-directed mutagenesis has been used to create a PstI site in the gene at the point encoding the signal peptide cleavage site. An internal methionine codon, the translation start for the cytoplasmic invertase, has been replaced by a serine codon. Mutants in the signal peptide sequence have been produced by replacing the region of the gene upstream of the PstI site with synthetic oligonucleotide cassettes with mixtures of nucleotides at several positions. The mutants could be divided into three classes based on their ability to secrete invertase. The first class of mutants produced secreted invertase, but in reduced amount. There is no obvious correlation between mutation and phenotype. The second class, represented by mutant 4-55B, also exhibited a reduced level of invertase, but a significant fraction (30%) of the enzyme is intracellular. This mutant had a delay in signal peptide cleavage which retards passage of invertase through the secretory pathway. The third class was defective in secretion. Most were defective in translocation from the cytoplasm to the lumen of the endoplasmic reticulum (ER), and produced enzymatically active, non-glycosylated pre-invertase in the cytoplasm. This class of mutant invertases, when transcribed and translated in vitro, was not processed by canine pancreas signal recognition particle (SRP) and microsomes. Comparison of the sequences of the mutant signal peptides of this non-translocating class identifies amino acids at the extreme amino-terminus as the causative defect.

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