Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.
We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae. The cDNA sequences encoding variant forms of influenza HA were expressed in S. cerevisiae. The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated. In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S. cerevisiae. The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery. The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S. cerevisiae compared with the signal-minus, nonglycosylated HA molecules. Although both the anchor-minus HA (HA500) and HA1 (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different. During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium. Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans. Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.
D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009975:Orthomyxoviridae / Q000235:genetics*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D021382:Protein Sorting Signals; D004247:DNA; D004262:DNA Restriction Enzymes