Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms.
The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation-specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.
Paterson
R G
RG
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
Lamb
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009975:Orthomyxoviridae / Q000201:enzymology / Q000235:genetics; D010252:Paramyxoviridae / Q000235:genetics; D010957:Plasmids; D011487:Protein Conformation; D055503:Protein Multimerization; D012995:Solubility; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D004273:DNA, Neoplasm; D046911:Macromolecular Substances; D008565:Membrane Proteins; D014760:Viral Fusion Proteins; D009439:Neuraminidase