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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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Mutant H1ΔLeu19  is expressed as two differently glycosylated forms. (A)  COS-7 cells were transfected  with cDNAs of H1, H1Δ,  H1Leu19, H1ΔLeu19, and  HC (the exoplasmic portion  of H1 equipped with the  cleavable signal sequence of  influenza virus hemagglutinin) as indicated. The transfected cells were labeled for  30 min with [35S]methionine,  solubilized, and subjected to  immunoprecipitation using  an antiserum directed against the COOH-terminal sequence of  H1. The immunoprecipitates were analyzed by gel electrophoresis and fluorography. (B) Immunoprecipitates were treated without (−) or with endoglycosidase H (E) before analysis by gel  electrophoresis. The positions of marker proteins are shown with  their molecular weights indicated in kD. The band indicated by  an asterisk represents a partially glycosylated species (see text).
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Figure 1: Mutant H1ΔLeu19 is expressed as two differently glycosylated forms. (A) COS-7 cells were transfected with cDNAs of H1, H1Δ, H1Leu19, H1ΔLeu19, and HC (the exoplasmic portion of H1 equipped with the cleavable signal sequence of influenza virus hemagglutinin) as indicated. The transfected cells were labeled for 30 min with [35S]methionine, solubilized, and subjected to immunoprecipitation using an antiserum directed against the COOH-terminal sequence of H1. The immunoprecipitates were analyzed by gel electrophoresis and fluorography. (B) Immunoprecipitates were treated without (−) or with endoglycosidase H (E) before analysis by gel electrophoresis. The positions of marker proteins are shown with their molecular weights indicated in kD. The band indicated by an asterisk represents a partially glycosylated species (see text).

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.


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)

Mutant H1ΔLeu19  is expressed as two differently glycosylated forms. (A)  COS-7 cells were transfected  with cDNAs of H1, H1Δ,  H1Leu19, H1ΔLeu19, and  HC (the exoplasmic portion  of H1 equipped with the  cleavable signal sequence of  influenza virus hemagglutinin) as indicated. The transfected cells were labeled for  30 min with [35S]methionine,  solubilized, and subjected to  immunoprecipitation using  an antiserum directed against the COOH-terminal sequence of  H1. The immunoprecipitates were analyzed by gel electrophoresis and fluorography. (B) Immunoprecipitates were treated without (−) or with endoglycosidase H (E) before analysis by gel  electrophoresis. The positions of marker proteins are shown with  their molecular weights indicated in kD. The band indicated by  an asterisk represents a partially glycosylated species (see text).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139883&req=5

Figure 1: Mutant H1ΔLeu19 is expressed as two differently glycosylated forms. (A) COS-7 cells were transfected with cDNAs of H1, H1Δ, H1Leu19, H1ΔLeu19, and HC (the exoplasmic portion of H1 equipped with the cleavable signal sequence of influenza virus hemagglutinin) as indicated. The transfected cells were labeled for 30 min with [35S]methionine, solubilized, and subjected to immunoprecipitation using an antiserum directed against the COOH-terminal sequence of H1. The immunoprecipitates were analyzed by gel electrophoresis and fluorography. (B) Immunoprecipitates were treated without (−) or with endoglycosidase H (E) before analysis by gel electrophoresis. The positions of marker proteins are shown with their molecular weights indicated in kD. The band indicated by an asterisk represents a partially glycosylated species (see text).
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.

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