Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain.
Bottom Line: Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones.The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive.In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.
Affiliation: Biozentrum, University of Basel, Switzerland.
The orientation of signal-anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH2-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7-25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.
Related in: MedlinePlus
Mentions: Expression of all the constructs with a truncated NH2terminal domain produced a small amount of protein of ∼33 kD, with an electrophoretic mobility intermediate between that of the twice glycosylated and the unglycosylated forms (indicated by asterisks in Figs. 1, 2, and 4). Upon endo H digestion, this material shifted to the position of the 30-kD unglycosylated form (Fig. 1 B); and in a protease protection assay, it was equally resistant as the twice glycosylated polypeptides (Fig. 2 B). These results indicate that the 33-kD species corresponds to type II polypeptides that were glycosylated only once. Incomplete glycosylation was generally not observed for constructs with the complete NH2-terminal domain of H1. Most likely, glycosylation at the site near the membrane (position 79 of the wild-type sequence) is slightly influenced by the presence or absence of the NH2-terminal domain.