MULTIPLE DETERMINANTS DIRECT THE ORIENTATION OF SIGNAL-ANCHOR PROTEINS - THE TOPOGENIC ROLE OF THE HYDROPHOBIC SIGNAL DOMAIN

The orientation of signal-anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the a polar, membrane-spanning domain and is influenced by the folding prope rties of the NH2-terminal sequence. However, these features are not ge nerally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutant s of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By rep lacing the 18-residue transmembrane segment of wild-type and mutant H1 by stretches of 7-25 leucine residues, we found that the length and h ydrophobicity of the apolar sequence significantly affected protein or ientation. Translocation of the NH:: terminus was favored by long, hyd rophobic sequences and translocation of the COOH terminus by short one s. The topogenic contributions of the transmembrane domain, the flanki ng charges, and a hydrophilic NH2-terminal portion were additive. In c ombination these determinants were sufficient to achieve unique membra ne insertion in either orientation.