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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: Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity.Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ~70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ~100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins.Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

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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: Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity.Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ~70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ~100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins.Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

View Article: PubMed Central - PubMed

Show MeSH
Related in: MedlinePlus