Limits...
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.

View Article: PubMed Central - PubMed

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4214783&req=5

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214783&req=5

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: 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