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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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N-terminal flanking residues help marginally hydrophobic NA TMDs to invert. (A) Logo plot displaying the conservation of the cytoplasmic-localized, N-terminal NA TMD flanking residues in the human H1N1 IAV sequences. (B) Diagram of the N-terminal mutations and deletions that were analyzed in the TMΔG+1.3NA76aa construct. (C) Immunoblots showing the orientation of the constructs depicted in B by the absence or presence of the expected two N-linked glycans (arrowheads). Cell lysates were harvested 48 h posttransfection, and a portion was treated with PNGase F before resolution by reducing Tris-tricine SDS–PAGE. (D) PM/IC ratios from cells expressing the constructs shown in B with representative cell section images. Insets, cellular localization of the constructs (green) with respect to the nucleus (blue).
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Figure 7: N-terminal flanking residues help marginally hydrophobic NA TMDs to invert. (A) Logo plot displaying the conservation of the cytoplasmic-localized, N-terminal NA TMD flanking residues in the human H1N1 IAV sequences. (B) Diagram of the N-terminal mutations and deletions that were analyzed in the TMΔG+1.3NA76aa construct. (C) Immunoblots showing the orientation of the constructs depicted in B by the absence or presence of the expected two N-linked glycans (arrowheads). Cell lysates were harvested 48 h posttransfection, and a portion was treated with PNGase F before resolution by reducing Tris-tricine SDS–PAGE. (D) PM/IC ratios from cells expressing the constructs shown in B with representative cell section images. Insets, cellular localization of the constructs (green) with respect to the nucleus (blue).

Mentions: The orientation of Nin-Cout (type II) TMDs can be influenced by the positioning of positively charged flanking residues and the length of the N-terminal flanking region (von Heijne, 1989; Kocik et al., 2012). In line with these findings, the 6-aa N-terminus is a highly conserved region in NA and includes a positively charged residue (K or, less commonly, R) adjacent to the TMD (Figure 7A). To investigate whether these N-terminal features aid the marginally hydrophobic NA TMD inversion, we examined the glycosylation pattern of the TM∆G +1.3NA76aa construct with several N-terminal deletions and mutations (Figure 7B).


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)

N-terminal flanking residues help marginally hydrophobic NA TMDs to invert. (A) Logo plot displaying the conservation of the cytoplasmic-localized, N-terminal NA TMD flanking residues in the human H1N1 IAV sequences. (B) Diagram of the N-terminal mutations and deletions that were analyzed in the TMΔG+1.3NA76aa construct. (C) Immunoblots showing the orientation of the constructs depicted in B by the absence or presence of the expected two N-linked glycans (arrowheads). Cell lysates were harvested 48 h posttransfection, and a portion was treated with PNGase F before resolution by reducing Tris-tricine SDS–PAGE. (D) PM/IC ratios from cells expressing the constructs shown in B with representative cell section images. Insets, cellular localization of the constructs (green) with respect to the nucleus (blue).
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Related In: Results  -  Collection

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Figure 7: N-terminal flanking residues help marginally hydrophobic NA TMDs to invert. (A) Logo plot displaying the conservation of the cytoplasmic-localized, N-terminal NA TMD flanking residues in the human H1N1 IAV sequences. (B) Diagram of the N-terminal mutations and deletions that were analyzed in the TMΔG+1.3NA76aa construct. (C) Immunoblots showing the orientation of the constructs depicted in B by the absence or presence of the expected two N-linked glycans (arrowheads). Cell lysates were harvested 48 h posttransfection, and a portion was treated with PNGase F before resolution by reducing Tris-tricine SDS–PAGE. (D) PM/IC ratios from cells expressing the constructs shown in B with representative cell section images. Insets, cellular localization of the constructs (green) with respect to the nucleus (blue).
Mentions: The orientation of Nin-Cout (type II) TMDs can be influenced by the positioning of positively charged flanking residues and the length of the N-terminal flanking region (von Heijne, 1989; Kocik et al., 2012). In line with these findings, the 6-aa N-terminus is a highly conserved region in NA and includes a positively charged residue (K or, less commonly, R) adjacent to the TMD (Figure 7A). To investigate whether these N-terminal features aid the marginally hydrophobic NA TMD inversion, we examined the glycosylation pattern of the TM∆G +1.3NA76aa construct with several N-terminal deletions and mutations (Figure 7B).

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