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Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.

Dou D, da Silva DV, Nordholm J, Wang H, Daniels R - Mol. Biol. Cell (2014)

Bottom Line: This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins.On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N(out)-C(in) HA and M2 TMDs but not the N(in)-C(out) TMDs from the type II membrane protein neuraminidase (NA).To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes.

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Inversion and PM localization of marginally hydrophobic NA TMDs is C-tail-length dependent. (A) PM/IC ratios of cells expressing the marginally hydrophobic NA construct (TM∆G +1.3NA) with increasing C-tail lengths and representative cell section images. (B) Orientation of the marginally hydrophobic NA TMD construct with increasing C-tail lengths was determined by the expected N-linked glycosylation pattern (depicted by the construct diagrams) at steady state (top, immunoblots) and after metabolic labeling (bottom, [35S]Met/Cys autoradiographs). Cell lysates were harvested 48 h posttransfection or after a 15-min pulse, and, where indicated, N-linked glycans were digested with PNGase F. The N-linked glycan number (arrowheads), partial PNGase F digestion (asterisks), and SDS-resistant TMD tetramer (Tet) are indicated. (C) The percent glycosylation of the marginally hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown. Glycosylation indicates that the constructs have the proper Nin-Cout orientation. (D) Analysis of the indicated 35S-labeled constructs by nonreducing and reducing SDS–PAGE reveals that only the glycosylated species form the expected intermolecular disulfide. Note the glycosylated-band molecular weight shifts ± DTT and the partial SDS-resistant tetrameric species.
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Figure 4: Inversion and PM localization of marginally hydrophobic NA TMDs is C-tail-length dependent. (A) PM/IC ratios of cells expressing the marginally hydrophobic NA construct (TM∆G +1.3NA) with increasing C-tail lengths and representative cell section images. (B) Orientation of the marginally hydrophobic NA TMD construct with increasing C-tail lengths was determined by the expected N-linked glycosylation pattern (depicted by the construct diagrams) at steady state (top, immunoblots) and after metabolic labeling (bottom, [35S]Met/Cys autoradiographs). Cell lysates were harvested 48 h posttransfection or after a 15-min pulse, and, where indicated, N-linked glycans were digested with PNGase F. The N-linked glycan number (arrowheads), partial PNGase F digestion (asterisks), and SDS-resistant TMD tetramer (Tet) are indicated. (C) The percent glycosylation of the marginally hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown. Glycosylation indicates that the constructs have the proper Nin-Cout orientation. (D) Analysis of the indicated 35S-labeled constructs by nonreducing and reducing SDS–PAGE reveals that only the glycosylated species form the expected intermolecular disulfide. Note the glycosylated-band molecular weight shifts ± DTT and the partial SDS-resistant tetrameric species.

Mentions: Our results thus far indicate that full-length NA with a marginally hydrophobic TMD localizes to the PM, likely because the longer C-tail facilitates inversion and potentially ER targeting. By making smaller increases in the C-tail length after the marginally hydrophobic NA TMD, we observed a stepwise increase in the average PM/IC ratios (Figure 4A). The increase slightly began with a C-tail length of 41 aa, which approximates the length of the ribosomal tunnel (Malkin and Rich, 1967; Blobel and Sabatini, 1970), and increased until a length of 76 aa, which showed a PM/IC profile distribution similar to the full-length construct (TM∆G +1.3NA440aa) in this steady-state assay. These results confirmed that the C-tail length contributes to PM localization of the marginally hydrophobic NA TMD.


Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.

Dou D, da Silva DV, Nordholm J, Wang H, Daniels R - Mol. Biol. Cell (2014)

Inversion and PM localization of marginally hydrophobic NA TMDs is C-tail-length dependent. (A) PM/IC ratios of cells expressing the marginally hydrophobic NA construct (TM∆G +1.3NA) with increasing C-tail lengths and representative cell section images. (B) Orientation of the marginally hydrophobic NA TMD construct with increasing C-tail lengths was determined by the expected N-linked glycosylation pattern (depicted by the construct diagrams) at steady state (top, immunoblots) and after metabolic labeling (bottom, [35S]Met/Cys autoradiographs). Cell lysates were harvested 48 h posttransfection or after a 15-min pulse, and, where indicated, N-linked glycans were digested with PNGase F. The N-linked glycan number (arrowheads), partial PNGase F digestion (asterisks), and SDS-resistant TMD tetramer (Tet) are indicated. (C) The percent glycosylation of the marginally hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown. Glycosylation indicates that the constructs have the proper Nin-Cout orientation. (D) Analysis of the indicated 35S-labeled constructs by nonreducing and reducing SDS–PAGE reveals that only the glycosylated species form the expected intermolecular disulfide. Note the glycosylated-band molecular weight shifts ± DTT and the partial SDS-resistant tetrameric species.
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Related In: Results  -  Collection

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Figure 4: Inversion and PM localization of marginally hydrophobic NA TMDs is C-tail-length dependent. (A) PM/IC ratios of cells expressing the marginally hydrophobic NA construct (TM∆G +1.3NA) with increasing C-tail lengths and representative cell section images. (B) Orientation of the marginally hydrophobic NA TMD construct with increasing C-tail lengths was determined by the expected N-linked glycosylation pattern (depicted by the construct diagrams) at steady state (top, immunoblots) and after metabolic labeling (bottom, [35S]Met/Cys autoradiographs). Cell lysates were harvested 48 h posttransfection or after a 15-min pulse, and, where indicated, N-linked glycans were digested with PNGase F. The N-linked glycan number (arrowheads), partial PNGase F digestion (asterisks), and SDS-resistant TMD tetramer (Tet) are indicated. (C) The percent glycosylation of the marginally hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown. Glycosylation indicates that the constructs have the proper Nin-Cout orientation. (D) Analysis of the indicated 35S-labeled constructs by nonreducing and reducing SDS–PAGE reveals that only the glycosylated species form the expected intermolecular disulfide. Note the glycosylated-band molecular weight shifts ± DTT and the partial SDS-resistant tetrameric species.
Mentions: Our results thus far indicate that full-length NA with a marginally hydrophobic TMD localizes to the PM, likely because the longer C-tail facilitates inversion and potentially ER targeting. By making smaller increases in the C-tail length after the marginally hydrophobic NA TMD, we observed a stepwise increase in the average PM/IC ratios (Figure 4A). The increase slightly began with a C-tail length of 41 aa, which approximates the length of the ribosomal tunnel (Malkin and Rich, 1967; Blobel and Sabatini, 1970), and increased until a length of 76 aa, which showed a PM/IC profile distribution similar to the full-length construct (TM∆G +1.3NA440aa) in this steady-state assay. These results confirmed that the C-tail length contributes to PM localization of the marginally hydrophobic NA TMD.

Bottom Line: This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins.On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N(out)-C(in) HA and M2 TMDs but not the N(in)-C(out) TMDs from the type II membrane protein neuraminidase (NA).To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes.

View Article: PubMed Central - PubMed

Show MeSH
Related in: MedlinePlus