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: The ASGP receptor subunit H1 is a type II membrane protein with an internal signal–anchor sequence. The hydrophobic core of the signal consists of 19 mostly apolar residues starting after arginine-40 and followed by 8 mainly polar but uncharged amino acids and two glutamic acid residues (positions 68 and 69). Upon transfection of H1 cDNA into COS-7 cells, 30 min labeling with [35S]methionine, and immunoprecipitation, a single polypeptide of 40 kD was recovered (Fig. 1 A, lane 1). Truncation of the NH2-terminal domain to only 4 residues in H1Δ resulted in a product of ∼36 kD (Fig. 1 A, lane 2) consistent with its reduced size. Digestion of an aliquot of the immunoprecipitates with endo H showed that H1Δ, like wild-type H1, was glycosylated at two sites in the COOH-terminal portion of the protein (Fig. 1 B, lanes 1–4; Beltzer et al., 1991). Deletion of the NH2-terminal portion thus had no effect on the topology of the protein in the membrane. This was to be expected according to the charge difference criterion (Hartmann et al., 1989): the charge difference between the 15 COOH-terminal and the 15 NH2-terminal flanking residues, Δ(C–N), was −3 for both H1 and H1Δ, statistically correlating with an Ncyt/Cexo orientation.