Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: Multiple intracellular and extracellular oligomeric and conformational forms

Reay G. Paterson*, Robert A. Lamb

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

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 influenza virus neuraminidase (NA) 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 ∼ 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.

Original languageEnglish (US)
Pages (from-to)999-1011
Number of pages13
JournalJournal of Cell Biology
Volume110
Issue number4
DOIs
StatePublished - Apr 1990

ASJC Scopus subject areas

  • Cell Biology

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