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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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Quantitation of the topology of the constructs H14Leu# and H1-4gLeu#. The insertion experiments including those  shown in Fig. 6 were quantified by densitometric scanning of the  fluorographs. The fraction of once glycosylated protein, i.e., with  Nexo/Ccyt orientation, is presented as percent of the total of all  forms as described in the legend to Fig. 5. The values for H14Leu# represent the mean of three or more experiments with  standard deviations; those for H1-4gLeu# represent single determinations performed simultaneously.
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Figure 7: Quantitation of the topology of the constructs H14Leu# and H1-4gLeu#. The insertion experiments including those shown in Fig. 6 were quantified by densitometric scanning of the fluorographs. The fraction of once glycosylated protein, i.e., with Nexo/Ccyt orientation, is presented as percent of the total of all forms as described in the legend to Fig. 5. The values for H14Leu# represent the mean of three or more experiments with standard deviations; those for H1-4gLeu# represent single determinations performed simultaneously.

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.


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)

Quantitation of the topology of the constructs H14Leu# and H1-4gLeu#. The insertion experiments including those  shown in Fig. 6 were quantified by densitometric scanning of the  fluorographs. The fraction of once glycosylated protein, i.e., with  Nexo/Ccyt orientation, is presented as percent of the total of all  forms as described in the legend to Fig. 5. The values for H14Leu# represent the mean of three or more experiments with  standard deviations; those for H1-4gLeu# represent single determinations performed simultaneously.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Quantitation of the topology of the constructs H14Leu# and H1-4gLeu#. The insertion experiments including those shown in Fig. 6 were quantified by densitometric scanning of the fluorographs. The fraction of once glycosylated protein, i.e., with Nexo/Ccyt orientation, is presented as percent of the total of all forms as described in the legend to Fig. 5. The values for H14Leu# represent the mean of three or more experiments with standard deviations; those for H1-4gLeu# represent single determinations performed simultaneously.
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.

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