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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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Marginally hydrophobic NA TMDs with a short C-tail can target to the ER cotranslationally. Ribosomally arrested chains with the indicated C-tail lengths and the marginally hydrophobic (A) and hydrophobic (B) NA TMD were in vitro translated in the absence and presence of rough ER microsomes (MS) before sedimentation through a sucrose cushion to isolate the microsomally associated chains (left). Representative experiments are shown for each construct, with the corresponding total amount of synthesized protein (Lysate) for the shortest constructs. To test for inversion and arrest, the glycosylation pattern of the 46-aa and 96-aa C-tail constructs were released with 1 mM puromycin (Puro) before sedimentation (right). The N-linked glycan number, unglycosylated chains (circles), and a potential SDS-resistant NA TMD dimer (asterisk) are indicated.
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Figure 6: Marginally hydrophobic NA TMDs with a short C-tail can target to the ER cotranslationally. Ribosomally arrested chains with the indicated C-tail lengths and the marginally hydrophobic (A) and hydrophobic (B) NA TMD were in vitro translated in the absence and presence of rough ER microsomes (MS) before sedimentation through a sucrose cushion to isolate the microsomally associated chains (left). Representative experiments are shown for each construct, with the corresponding total amount of synthesized protein (Lysate) for the shortest constructs. To test for inversion and arrest, the glycosylation pattern of the 46-aa and 96-aa C-tail constructs were released with 1 mM puromycin (Puro) before sedimentation (right). The N-linked glycan number, unglycosylated chains (circles), and a potential SDS-resistant NA TMD dimer (asterisk) are indicated.

Mentions: In addition to affecting membrane integration and orientation, decreased hydrophobicity in Nin-Cout (type II) TMDs and shorter C-tail length could also potentially affect their SRP recognition and hence their ER targeting capability (Bird et al., 1990; Hatsuzawa et al., 1997). Therefore we created a series of ribosomally arrested chains with elongated C-tails using the hydrophobic and marginally hydrophobic NA TMDs and assayed their ability to direct targeting to rough ER microsomes. As expected, no targeting to the ER microsomes was observed when the marginally hydrophobic TMD was largely sequestered in the ribosome with a 22-aa C-tail (Figure 6A). With a 36-aa C-tail, a higher proportion of the marginally hydrophobic NA TMD ribosomally arrested chains sedimented with the ER microsomes, and the proportion substantially increased with a 46-aa C-tail, which is similar to the 49-aa C-tail on the SRP-dependent M2 TMD (Hull et al., 1988). A comparable but more efficient ER targeting profile was observed with the hydrophobic NA TMD ribosomally arrested chains (Figure 6B).


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)

Marginally hydrophobic NA TMDs with a short C-tail can target to the ER cotranslationally. Ribosomally arrested chains with the indicated C-tail lengths and the marginally hydrophobic (A) and hydrophobic (B) NA TMD were in vitro translated in the absence and presence of rough ER microsomes (MS) before sedimentation through a sucrose cushion to isolate the microsomally associated chains (left). Representative experiments are shown for each construct, with the corresponding total amount of synthesized protein (Lysate) for the shortest constructs. To test for inversion and arrest, the glycosylation pattern of the 46-aa and 96-aa C-tail constructs were released with 1 mM puromycin (Puro) before sedimentation (right). The N-linked glycan number, unglycosylated chains (circles), and a potential SDS-resistant NA TMD dimer (asterisk) are indicated.
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Figure 6: Marginally hydrophobic NA TMDs with a short C-tail can target to the ER cotranslationally. Ribosomally arrested chains with the indicated C-tail lengths and the marginally hydrophobic (A) and hydrophobic (B) NA TMD were in vitro translated in the absence and presence of rough ER microsomes (MS) before sedimentation through a sucrose cushion to isolate the microsomally associated chains (left). Representative experiments are shown for each construct, with the corresponding total amount of synthesized protein (Lysate) for the shortest constructs. To test for inversion and arrest, the glycosylation pattern of the 46-aa and 96-aa C-tail constructs were released with 1 mM puromycin (Puro) before sedimentation (right). The N-linked glycan number, unglycosylated chains (circles), and a potential SDS-resistant NA TMD dimer (asterisk) are indicated.
Mentions: In addition to affecting membrane integration and orientation, decreased hydrophobicity in Nin-Cout (type II) TMDs and shorter C-tail length could also potentially affect their SRP recognition and hence their ER targeting capability (Bird et al., 1990; Hatsuzawa et al., 1997). Therefore we created a series of ribosomally arrested chains with elongated C-tails using the hydrophobic and marginally hydrophobic NA TMDs and assayed their ability to direct targeting to rough ER microsomes. As expected, no targeting to the ER microsomes was observed when the marginally hydrophobic TMD was largely sequestered in the ribosome with a 22-aa C-tail (Figure 6A). With a 36-aa C-tail, a higher proportion of the marginally hydrophobic NA TMD ribosomally arrested chains sedimented with the ER microsomes, and the proportion substantially increased with a 46-aa C-tail, which is similar to the 49-aa C-tail on the SRP-dependent M2 TMD (Hull et al., 1988). A comparable but more efficient ER targeting profile was observed with the hydrophobic NA TMD ribosomally arrested chains (Figure 6B).

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