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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 mislocalize in cells. (A) Cell PM/IC ratios for the full-length Nin-Cout membrane protein NA440aa with a hydrophobic (ΔGapp = −0.7 kcal/mol) and a marginally hydrophobic (ΔGapp = +1.3 kcal/mol) TMD, as well as the Nout-Cin membrane protein M270aa with a hydrophobic (ΔGapp = −1.1 kcal/mol) TMD and the intracellular (IC) marker. Right, representative cell section images. (B) PM/IC ratios from cells expressing these membrane proteins with a C-tail truncated to 36 aa. Right, representative images. (C) Higher-magnification image showing localization of TM∆G +1.3NA36aa (white) in “ring-like” vesicle structures near the nucleus (blue). (D) Vesicles from cells transfected with the indicated constructs were separated from the cytoplasm (Cyto) and subjected to an alkaline extraction to separate integral (P) from peripheral (SN) membrane proteins. Representative immunoblots with GFP as the soluble cytoplasm control. (E) Vesicle and cytoplasmic fractions from transfected cells were analyzed by flotation on sucrose cushions of different density. The dot blot shows the intensity of TM∆G +1.3NA36aa and TM∆G +1.3NA440aa that floated (SN) or pelleted (P) at each density, and the enzymatic activity distribution for TM∆G +1.3NA440aa provides an added control. (F) Similar analysis as in E, except that TM∆G +1.3NA36aa was radiolabeled for 30 min and isolated after the separation using Ni-Sepharose before resolution by SDS–PAGE.
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Figure 2: Marginally hydrophobic NA TMDs with a short C-tail mislocalize in cells. (A) Cell PM/IC ratios for the full-length Nin-Cout membrane protein NA440aa with a hydrophobic (ΔGapp = −0.7 kcal/mol) and a marginally hydrophobic (ΔGapp = +1.3 kcal/mol) TMD, as well as the Nout-Cin membrane protein M270aa with a hydrophobic (ΔGapp = −1.1 kcal/mol) TMD and the intracellular (IC) marker. Right, representative cell section images. (B) PM/IC ratios from cells expressing these membrane proteins with a C-tail truncated to 36 aa. Right, representative images. (C) Higher-magnification image showing localization of TM∆G +1.3NA36aa (white) in “ring-like” vesicle structures near the nucleus (blue). (D) Vesicles from cells transfected with the indicated constructs were separated from the cytoplasm (Cyto) and subjected to an alkaline extraction to separate integral (P) from peripheral (SN) membrane proteins. Representative immunoblots with GFP as the soluble cytoplasm control. (E) Vesicle and cytoplasmic fractions from transfected cells were analyzed by flotation on sucrose cushions of different density. The dot blot shows the intensity of TM∆G +1.3NA36aa and TM∆G +1.3NA440aa that floated (SN) or pelleted (P) at each density, and the enzymatic activity distribution for TM∆G +1.3NA440aa provides an added control. (F) Similar analysis as in E, except that TM∆G +1.3NA36aa was radiolabeled for 30 min and isolated after the separation using Ni-Sepharose before resolution by SDS–PAGE.

Mentions: This approach was first applied to evaluate the localization of full-length NA with natural hydrophobic (ΔGapp = −0.7 kcal/mol) and marginally hydrophobic (ΔGapp = +1.3) Nin-Cout TMDs. Including the epitope tag, both constructs possess a long, 440-aa C-tail that follows the TMD, and from here on are referred to by the nomenclature TM∆G XNAZaa, where X is the predicted TMD hydrophobicity (ΔGapp value in kcal/mol) and Z is the C-tail length in amino acids (aa). As expected, both full-length NA constructs (TM∆G −0.7NA440aa and TM∆G +1.3NA440aa) localized to the PM with an average PM/IC ratio >2 (Figure 2A). Similarly, full-length M2 with a hydrophobic TMD (ΔGapp = −1.1 kcal/mol) and a C-tail of 70 aa (including the epitope tag) also localized to the PM.


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 mislocalize in cells. (A) Cell PM/IC ratios for the full-length Nin-Cout membrane protein NA440aa with a hydrophobic (ΔGapp = −0.7 kcal/mol) and a marginally hydrophobic (ΔGapp = +1.3 kcal/mol) TMD, as well as the Nout-Cin membrane protein M270aa with a hydrophobic (ΔGapp = −1.1 kcal/mol) TMD and the intracellular (IC) marker. Right, representative cell section images. (B) PM/IC ratios from cells expressing these membrane proteins with a C-tail truncated to 36 aa. Right, representative images. (C) Higher-magnification image showing localization of TM∆G +1.3NA36aa (white) in “ring-like” vesicle structures near the nucleus (blue). (D) Vesicles from cells transfected with the indicated constructs were separated from the cytoplasm (Cyto) and subjected to an alkaline extraction to separate integral (P) from peripheral (SN) membrane proteins. Representative immunoblots with GFP as the soluble cytoplasm control. (E) Vesicle and cytoplasmic fractions from transfected cells were analyzed by flotation on sucrose cushions of different density. The dot blot shows the intensity of TM∆G +1.3NA36aa and TM∆G +1.3NA440aa that floated (SN) or pelleted (P) at each density, and the enzymatic activity distribution for TM∆G +1.3NA440aa provides an added control. (F) Similar analysis as in E, except that TM∆G +1.3NA36aa was radiolabeled for 30 min and isolated after the separation using Ni-Sepharose before resolution by SDS–PAGE.
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Figure 2: Marginally hydrophobic NA TMDs with a short C-tail mislocalize in cells. (A) Cell PM/IC ratios for the full-length Nin-Cout membrane protein NA440aa with a hydrophobic (ΔGapp = −0.7 kcal/mol) and a marginally hydrophobic (ΔGapp = +1.3 kcal/mol) TMD, as well as the Nout-Cin membrane protein M270aa with a hydrophobic (ΔGapp = −1.1 kcal/mol) TMD and the intracellular (IC) marker. Right, representative cell section images. (B) PM/IC ratios from cells expressing these membrane proteins with a C-tail truncated to 36 aa. Right, representative images. (C) Higher-magnification image showing localization of TM∆G +1.3NA36aa (white) in “ring-like” vesicle structures near the nucleus (blue). (D) Vesicles from cells transfected with the indicated constructs were separated from the cytoplasm (Cyto) and subjected to an alkaline extraction to separate integral (P) from peripheral (SN) membrane proteins. Representative immunoblots with GFP as the soluble cytoplasm control. (E) Vesicle and cytoplasmic fractions from transfected cells were analyzed by flotation on sucrose cushions of different density. The dot blot shows the intensity of TM∆G +1.3NA36aa and TM∆G +1.3NA440aa that floated (SN) or pelleted (P) at each density, and the enzymatic activity distribution for TM∆G +1.3NA440aa provides an added control. (F) Similar analysis as in E, except that TM∆G +1.3NA36aa was radiolabeled for 30 min and isolated after the separation using Ni-Sepharose before resolution by SDS–PAGE.
Mentions: This approach was first applied to evaluate the localization of full-length NA with natural hydrophobic (ΔGapp = −0.7 kcal/mol) and marginally hydrophobic (ΔGapp = +1.3) Nin-Cout TMDs. Including the epitope tag, both constructs possess a long, 440-aa C-tail that follows the TMD, and from here on are referred to by the nomenclature TM∆G XNAZaa, where X is the predicted TMD hydrophobicity (ΔGapp value in kcal/mol) and Z is the C-tail length in amino acids (aa). As expected, both full-length NA constructs (TM∆G −0.7NA440aa and TM∆G +1.3NA440aa) localized to the PM with an average PM/IC ratio >2 (Figure 2A). Similarly, full-length M2 with a hydrophobic TMD (ΔGapp = −1.1 kcal/mol) and a C-tail of 70 aa (including the epitope tag) also localized to the PM.

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