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Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain.

Wahlberg JM, Spiess M - J. Cell Biol. (1997)

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.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Switzerland.

ABSTRACT
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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Amino acid sequence of the signal–anchor domain of  H1 mutant constructs. The hydrophobic transmembrane segments  and their flanking sequences are listed. H1-4g and H1-4gLeu# are  identical to H1-4 and H1-4Leu# except for the insertion of the  tripeptide sequence MTM following asparagine-13 in the NH2terminal portion, which creates a potential glycosylation site.
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Figure 3: Amino acid sequence of the signal–anchor domain of H1 mutant constructs. The hydrophobic transmembrane segments and their flanking sequences are listed. H1-4g and H1-4gLeu# are identical to H1-4 and H1-4Leu# except for the insertion of the tripeptide sequence MTM following asparagine-13 in the NH2terminal portion, which creates a potential glycosylation site.

Mentions: Replacing the transmembrane domain of H1 by a sequence of 19 leucine residues increased its hydrophobicity without altering the physical length of the domain. To further explore the topogenic role of the hydrophobic domain, we prepared mutant constructs of H1 and H1Δ with artificial transmembrane domains consisting of 7–25 leucine residues. Thus the overall hydrophobicity and the length of the apolar domain were varied in parallel (Fig. 3).


Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain.

Wahlberg JM, Spiess M - J. Cell Biol. (1997)

Amino acid sequence of the signal–anchor domain of  H1 mutant constructs. The hydrophobic transmembrane segments  and their flanking sequences are listed. H1-4g and H1-4gLeu# are  identical to H1-4 and H1-4Leu# except for the insertion of the  tripeptide sequence MTM following asparagine-13 in the NH2terminal portion, which creates a potential glycosylation site.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139883&req=5

Figure 3: Amino acid sequence of the signal–anchor domain of H1 mutant constructs. The hydrophobic transmembrane segments and their flanking sequences are listed. H1-4g and H1-4gLeu# are identical to H1-4 and H1-4Leu# except for the insertion of the tripeptide sequence MTM following asparagine-13 in the NH2terminal portion, which creates a potential glycosylation site.
Mentions: Replacing the transmembrane domain of H1 by a sequence of 19 leucine residues increased its hydrophobicity without altering the physical length of the domain. To further explore the topogenic role of the hydrophobic domain, we prepared mutant constructs of H1 and H1Δ with artificial transmembrane domains consisting of 7–25 leucine residues. Thus the overall hydrophobicity and the length of the apolar domain were varied in parallel (Fig. 3).

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.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Switzerland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus