Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.
Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002458:Cell Fractionation; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D006026:Glycoside Hydrolases / Q000235:genetics*; D006031:Glycosylation; D008970:Molecular Weight; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D043324:beta-Fructofuranosidase
D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase