Isolation and Characterization of the Hydrophobic COOH-terminal Domains of the Sindbis Virion Glycoproteins

Abstract

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from El and E2, respectively). Amino acid composition data and NH_2-terminal sequence analysis established their location at the extreme COOR-terminal end of each glycoprotein. REI and RE2 are rich in hydrophobic amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with El and E2 was found to be clustered in their hydrophobic. membrane-associated roots. Beginning six to seven residues from their NH2 termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), El and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, El probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOR-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOR-terminal end of E2, which is protected by the virion envelope from digestion by α-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.