The length requirement for a functional uncleaved signal/anchor (S/A) domain of the paramyxovirus hemagglutinin-neuraminidase (HN) type II glycoprotein was analyzed. HN mutants with progressive NH-terminal S/A deletions or insertions were expressed in HeLa cells, and the membrane targeting, folding, tetramer assembly, and intracellular transport of the proteins were examined. Changing the length of the S/A by two residues resulted in HN mutants that displayed aberrant endoplasmic reticulum (ER) membrane targeting or translocation. This phenotype did not simply reflect upper or lower limitations on the size of a functional S/A, because normal signaling was restored by further alterations involving three or four residues. Likewise, ER-to-Golgi transport of mutants containing deletions of one or two S/A residues was delayed (30% of WT) or blocked, but transport was restored for a mutant with a total of three deleted residues. HN mutants with S/A insertions of three or four Leu residues differed from wild-type HN by having heterogeneous Golgi-specific carbohydrate modifications. Differences in ER-to-Golgi transport of the mutants did not strictly correlate with defects in either native folding of the ectodomain or the assembly of two dimers into a tetramer. Together, these data suggest that efficient entry into and exit from the ER are sensitive to changes in the HN S/A that may reflect alterations to a structural requirement along one side of an -helix.

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