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: As is shown in Fig. 6 A and quantified in Fig. 7 (open squares), transmembrane segments >13 leucine residues showed increased translocation of the NH2 terminus and were thus topogenically active even in an internal position. Virtually unique type III insertion was observed for H14Leu22 and H1-4Leu25. Whereas the longer leucine sequences favored type III insertion, shorter ones did not shift the balance towards type II insertion. H1-4Leu13, H14Leu10, and H1-4Leu7 did not insert as type II proteins to any larger extent than H1-4. Shortening the oligo–leucine domain more effectively increased the fraction of Ncyt/Cexo polypeptides in constructs with the signal domain located close to the NH2 terminus than further inside the protein.