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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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An elongated C-tail can invert the Nout-Cin M2 TMD and is associated with marginally hydrophobic human Nin-Cout (type II) TMDs. (A) Immunoblot of reduced and nonreduced cell lysates showing the Nout-Cin orientation of M2 based on the intermolecular disulfide bonds formed by its N-terminal Cys residues in the ER lumen. (B) Immunoblots of untreated and PNGase F–treated lysates showing the glycosylation patterns for TMΔG+1.3NA76aa, M2 with the 76-aa NA C-tail, full-length NA (TMΔG+1.3NA440aa), and M2 with the full-length 440-aa NA C-tail with the enzymatic domain. The number of N-linked glycans for each species is indicated. (C) Enzymatic activity was used to confirm M2 TMD inversion, as NA only folds within the ER lumen. The activity rate of M2 with the full-length 440-aa NA C-tail was calculated in comparison to lysates from cells expressing full-length NA as described in da Silva et al. (2013). (D) TMD hydrophobicity for the annotated type II human membrane proteins with respect to the length of the C-terminus after their TMD (C-tail). Dashed line at 100 aa corresponds to the ∼50% inversion point for newly synthesized NA with a marginally hydrophobic TMD. Regions covering the marginally hydrophobic TMDs and potential tail-anchored pathway substrates are highlighted. Protein sequences were obtained from Uniprot and analyzed using MPEx. The raw data and references are given in Supplemental Table S1.
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Figure 8: An elongated C-tail can invert the Nout-Cin M2 TMD and is associated with marginally hydrophobic human Nin-Cout (type II) TMDs. (A) Immunoblot of reduced and nonreduced cell lysates showing the Nout-Cin orientation of M2 based on the intermolecular disulfide bonds formed by its N-terminal Cys residues in the ER lumen. (B) Immunoblots of untreated and PNGase F–treated lysates showing the glycosylation patterns for TMΔG+1.3NA76aa, M2 with the 76-aa NA C-tail, full-length NA (TMΔG+1.3NA440aa), and M2 with the full-length 440-aa NA C-tail with the enzymatic domain. The number of N-linked glycans for each species is indicated. (C) Enzymatic activity was used to confirm M2 TMD inversion, as NA only folds within the ER lumen. The activity rate of M2 with the full-length 440-aa NA C-tail was calculated in comparison to lysates from cells expressing full-length NA as described in da Silva et al. (2013). (D) TMD hydrophobicity for the annotated type II human membrane proteins with respect to the length of the C-terminus after their TMD (C-tail). Dashed line at 100 aa corresponds to the ∼50% inversion point for newly synthesized NA with a marginally hydrophobic TMD. Regions covering the marginally hydrophobic TMDs and potential tail-anchored pathway substrates are highlighted. Protein sequences were obtained from Uniprot and analyzed using MPEx. The raw data and references are given in Supplemental Table S1.

Mentions: To examine whether the cotranslational integration process contributes to TMD inversion more directly, we tested whether elongating the C-tail following the Nout-Cin M2 TMD could facilitate its inversion. As shown by the oxidation of M2 into homodimers and tetramers via its N-terminal Cys residues, full-length M2 with a 70-aa C-tail (including the epitope tag) possessed an Nout-Cin orientation at steady-state (Figure 8A). Similarly, when the 76-aa NA C-tail was fused to the M2 N-terminus and TMD, the Nout-Cin orientation remained unchanged, as no glycosylation of the NA C-tail was observed (Figure 8B, lanes 3 and 4). However, when the full-length 440-aa NA C-tail was fused to the M2 N-terminus and TMD, a reasonable amount of inversion to an Nin-Cout orientation occurred, based on glycosylation (Figure 8B, lanes 7 and 8) and the production of enzymatically active NA (Figure 8C). The ability to invert the hydrophobic M2 TMD in the absence of its single C-terminal positive charge upon substantially lengthening the 76-aa NA C-tail to 440 aa indicates that cotranslational synthesis and/or integration contributes to the inversion process. However, the production of mixed orientated species also implies that TMDs likely possess their own topology determinants in addition to their cytoplasmic-localized flanking residues.


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)

An elongated C-tail can invert the Nout-Cin M2 TMD and is associated with marginally hydrophobic human Nin-Cout (type II) TMDs. (A) Immunoblot of reduced and nonreduced cell lysates showing the Nout-Cin orientation of M2 based on the intermolecular disulfide bonds formed by its N-terminal Cys residues in the ER lumen. (B) Immunoblots of untreated and PNGase F–treated lysates showing the glycosylation patterns for TMΔG+1.3NA76aa, M2 with the 76-aa NA C-tail, full-length NA (TMΔG+1.3NA440aa), and M2 with the full-length 440-aa NA C-tail with the enzymatic domain. The number of N-linked glycans for each species is indicated. (C) Enzymatic activity was used to confirm M2 TMD inversion, as NA only folds within the ER lumen. The activity rate of M2 with the full-length 440-aa NA C-tail was calculated in comparison to lysates from cells expressing full-length NA as described in da Silva et al. (2013). (D) TMD hydrophobicity for the annotated type II human membrane proteins with respect to the length of the C-terminus after their TMD (C-tail). Dashed line at 100 aa corresponds to the ∼50% inversion point for newly synthesized NA with a marginally hydrophobic TMD. Regions covering the marginally hydrophobic TMDs and potential tail-anchored pathway substrates are highlighted. Protein sequences were obtained from Uniprot and analyzed using MPEx. The raw data and references are given in Supplemental Table S1.
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Related In: Results  -  Collection

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Figure 8: An elongated C-tail can invert the Nout-Cin M2 TMD and is associated with marginally hydrophobic human Nin-Cout (type II) TMDs. (A) Immunoblot of reduced and nonreduced cell lysates showing the Nout-Cin orientation of M2 based on the intermolecular disulfide bonds formed by its N-terminal Cys residues in the ER lumen. (B) Immunoblots of untreated and PNGase F–treated lysates showing the glycosylation patterns for TMΔG+1.3NA76aa, M2 with the 76-aa NA C-tail, full-length NA (TMΔG+1.3NA440aa), and M2 with the full-length 440-aa NA C-tail with the enzymatic domain. The number of N-linked glycans for each species is indicated. (C) Enzymatic activity was used to confirm M2 TMD inversion, as NA only folds within the ER lumen. The activity rate of M2 with the full-length 440-aa NA C-tail was calculated in comparison to lysates from cells expressing full-length NA as described in da Silva et al. (2013). (D) TMD hydrophobicity for the annotated type II human membrane proteins with respect to the length of the C-terminus after their TMD (C-tail). Dashed line at 100 aa corresponds to the ∼50% inversion point for newly synthesized NA with a marginally hydrophobic TMD. Regions covering the marginally hydrophobic TMDs and potential tail-anchored pathway substrates are highlighted. Protein sequences were obtained from Uniprot and analyzed using MPEx. The raw data and references are given in Supplemental Table S1.
Mentions: To examine whether the cotranslational integration process contributes to TMD inversion more directly, we tested whether elongating the C-tail following the Nout-Cin M2 TMD could facilitate its inversion. As shown by the oxidation of M2 into homodimers and tetramers via its N-terminal Cys residues, full-length M2 with a 70-aa C-tail (including the epitope tag) possessed an Nout-Cin orientation at steady-state (Figure 8A). Similarly, when the 76-aa NA C-tail was fused to the M2 N-terminus and TMD, the Nout-Cin orientation remained unchanged, as no glycosylation of the NA C-tail was observed (Figure 8B, lanes 3 and 4). However, when the full-length 440-aa NA C-tail was fused to the M2 N-terminus and TMD, a reasonable amount of inversion to an Nin-Cout orientation occurred, based on glycosylation (Figure 8B, lanes 7 and 8) and the production of enzymatically active NA (Figure 8C). The ability to invert the hydrophobic M2 TMD in the absence of its single C-terminal positive charge upon substantially lengthening the 76-aa NA C-tail to 440 aa indicates that cotranslational synthesis and/or integration contributes to the inversion process. However, the production of mixed orientated species also implies that TMDs likely possess their own topology determinants in addition to their cytoplasmic-localized flanking residues.

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