Deletion mutation in the signal anchor domain activates cleavage of the influenza virus neuraminidase, a type II transmembrane protein.
Influenza virus neuraminidase (NA) is a type II integral membrane protein with a long hydrophobic domain [29 amino acids (aa)] at the N terminus that functions as an uncleaved signal for translocation into the endoplasmic reticulum and anchors the protein in the membrane. The function of the transmembrane domain in intracellular transport was investigated by deletion mutagenesis. Expression of the mutated NA in eukaryotic cells and by in vitro translation in the presence of membranes showed that the deletion of eight amino acids (aa 28 to 35) from the carboxy end of the signal anchor domain resulted in cleavage, probably by the signal peptidase and secretion of NA into the culture medium. The mutant NA (N28-35) was present inside the cell predominantly as dimers, secreted as dimers, and was enzymatically inactive. When translated in vitro in the presence of dog pancreatic microsomal membranes, the N28-35 protein underwent cleavage and did not remain anchored to membranes. Two other deletion mutants in the transmembrane domain, N7-17 and N17-23, were partially cleaved and secreted, whereas two mutants, one (N19-27) lacking nine aa in the central region and the other (N1-14) lacking the first 14aa from the N terminus remained uncleaved and exhibited a phenotype similar to the wild-type NA. We conclude that the longer transmembrane domain (29 aa) may play an important role in determining that type II signal is not cleaved during translocation; however, in addition, adjacent amino acid sequences also provide determinants important in signal cleavage.
Hogue
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BG
Department of Microbiology and Immunology, Jonsson Comprehensive Cancer Center, University of California, Los Angeles School of Medicine 90024-1747.
Nayak
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002474:Cell-Free System; D002478:Cells, Cultured; D006224:Cricetinae; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D009980:Influenza A virus / Q000201:enzymology*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
D021382:Protein Sorting Signals; D009439:Neuraminidase