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: In the constructs H1Leu#, described above, the leucine sequences had no effect on the topology of the proteins in the ER membrane. This could be due to the charge difference of −4.5 (as calculated according to Hartmann et al., 1989), which is more negative than in the H1ΔLeu# series. In addition, the presence of a sizable NH2-terminal hydrophilic domain is likely to disfavor Nexo/Ccyt insertion. To test whether a long, hydrophobic signal domain in an internal position can influence insertion at all, we used H1-4, a previously described mutant of H1 in which the two NH2-terminal and the two COOH-terminal charges flanking the transmembrane domain had been mutated to residues of opposite charge (Beltzer et al., 1991; Fig. 3). Even though the charge difference of this mutant was strongly positive (+5), only half of the polypeptides were inserted as type III proteins (Fig. 6 A, lane 1). In this mutant construct, the 19 hydrophobic amino acids of the signal anchor were replaced by 7–25 consecutive leucine residues, thus generating the mutant series H1-4Leu#.