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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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Hydrophobic NA TMDs are less dependent on C-tail length for inversion. (A) The orientation of the hydrophobic NA TMD construct with increasing C-tail lengths was analyzed at steady-state (top, immunoblots) and after metabolic labeling for 15 min (bottom, [35S]Met/Cys autoradiographs) as described in Figure 4B. The N-linked glycan number (arrowheads) and partial PNGase F digested species (asterisk) are shown. (B) The percent glycosylation observed for the hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown.
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Figure 5: Hydrophobic NA TMDs are less dependent on C-tail length for inversion. (A) The orientation of the hydrophobic NA TMD construct with increasing C-tail lengths was analyzed at steady-state (top, immunoblots) and after metabolic labeling for 15 min (bottom, [35S]Met/Cys autoradiographs) as described in Figure 4B. The N-linked glycan number (arrowheads) and partial PNGase F digested species (asterisk) are shown. (B) The percent glycosylation observed for the hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown.

Mentions: Because the hydrophobic NA TMDs (ΔGapp < 0 kcal/mol) with short, 36-aa C-tails properly localized to the PM (Figures 2 and 3), we also examined the hydrophobic NA TMD orientation with increasing C-tail lengths. At steady-state, these constructs were already ∼65% glycosylated, with a C-tail of 36 aa, and their glycosylation percentage increased further once the C-tail length was >61 aa (Figure 5A, top, immunoblots, and B). The analysis of the nascent chains after a 15-min pulse showed that the percentage of glycosylated species increased with the C-tail length beginning from 36 aa (Figure 5A, bottom, audioradiographs, and B). These results demonstrate that hydrophobic NA TMDs benefit from, but do not require, the cotranslational membrane insertion process (ribosomal attachment and/or synthesis) for their inversion, which explains their PM localization with short C-tails.


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)

Hydrophobic NA TMDs are less dependent on C-tail length for inversion. (A) The orientation of the hydrophobic NA TMD construct with increasing C-tail lengths was analyzed at steady-state (top, immunoblots) and after metabolic labeling for 15 min (bottom, [35S]Met/Cys autoradiographs) as described in Figure 4B. The N-linked glycan number (arrowheads) and partial PNGase F digested species (asterisk) are shown. (B) The percent glycosylation observed for the hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown.
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Related In: Results  -  Collection

Show All Figures
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Figure 5: Hydrophobic NA TMDs are less dependent on C-tail length for inversion. (A) The orientation of the hydrophobic NA TMD construct with increasing C-tail lengths was analyzed at steady-state (top, immunoblots) and after metabolic labeling for 15 min (bottom, [35S]Met/Cys autoradiographs) as described in Figure 4B. The N-linked glycan number (arrowheads) and partial PNGase F digested species (asterisk) are shown. (B) The percent glycosylation observed for the hydrophobic NA TMDs with respect to C-tail length from steady-state and 35S-labeling experiments is shown.
Mentions: Because the hydrophobic NA TMDs (ΔGapp < 0 kcal/mol) with short, 36-aa C-tails properly localized to the PM (Figures 2 and 3), we also examined the hydrophobic NA TMD orientation with increasing C-tail lengths. At steady-state, these constructs were already ∼65% glycosylated, with a C-tail of 36 aa, and their glycosylation percentage increased further once the C-tail length was >61 aa (Figure 5A, top, immunoblots, and B). The analysis of the nascent chains after a 15-min pulse showed that the percentage of glycosylated species increased with the C-tail length beginning from 36 aa (Figure 5A, bottom, audioradiographs, and B). These results demonstrate that hydrophobic NA TMDs benefit from, but do not require, the cotranslational membrane insertion process (ribosomal attachment and/or synthesis) for their inversion, which explains their PM localization with short C-tails.

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