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.
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Mentions: Lack of glycosylation could be due to a reduced efficiency of the mutated signal sequence in targeting the protein to the ER membrane, resulting in a soluble, cytosolic polypeptide. Alternatively, all products may still be integrated in the membrane, but some with the opposite Nexo/ Ccyt orientation leaving the COOH-terminal portion with the glycosylation sites in the cytosol. Membrane association of the unglycosylated form was analyzed by saponin and alkaline extraction of expressing COS-7 cells. After labeling with [35S]methionine, the cells were incubated with 0.1% saponin for 30 min at 4°C to release soluble proteins into the supernatant. Extracted and membrane-associated proteins were then immunoprecipitated from the saponin extract (S) and from the residual cells (C), respectively (Fig. 2 A). A secretory form of the exoplasmic portion of H1 with a cleavable signal sequence (HC) was efficiently extracted (Fig. 2 A, lanes 10–12), whereas wild-type H1 and H1Δ were completely retained with the cellular membranes (Fig. 1 A, lanes 1–6). Both the glycosylated and the unglycosylated forms of H1ΔLeu were clearly membrane associated in this assay (Fig. 1 A, lanes 7–9). For alkaline extraction, the transfected and labeled cells were homogenized, exposed to pH 11.5, and then separated into soluble fraction (S) and membrane pellet (P) by centrifugation through a sucrose cushion (Fig. 2 B). Like H1Δ (Fig. 2 B, lanes 4–6), both forms of H1ΔLeu19 were recovered in the pellet fraction (Fig. 2 B, lanes 7–9). These results indicate that the unglycosylated form of H1ΔLeu19 is integrated in the membrane and argues against a reduced ability of the Leu19 domain to function as a signal sequence.