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A membrane topology model for human interferon inducible transmembrane protein 1.

Weston S, Czieso S, White IJ, Smith SE, Kellam P, Marsh M - PLoS ONE (2014)

Bottom Line: Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular.Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins.This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.

ABSTRACT
InterFeron Inducible TransMembrane proteins 1-3 (IFITM1, IFITM2 and IFITM3) are a family of proteins capable of inhibiting the cellular entry of numerous human and animal viruses. IFITM1-3 are unique amongst the currently described viral restriction factors in their apparent ability to block viral entry. This restrictive property is dependant on the localisation of the proteins to plasma and endosomal membranes, which constitute the main portals of viral entry into cells. The topology of the IFITM proteins within cell membranes is an unresolved aspect of their biology. Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular. Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins. This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

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Related in: MedlinePlus

Co-staining with N- and C-terminal antibodies.Permeabilised IFITM2-HA (A) and IFITM3-HA (B) expressing cells were stained with antibodies against the C-terminal HA-tag (green [Alexa-488]) and the NTD, using the anti-IFITM3-NTD antibody (red [Alexa-647]). Images are single optical sections (0.25 µm thick) through the cells. Scale bars represent 15 µm. See also Table 2 and 3 for image analysis.
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pone-0104341-g009: Co-staining with N- and C-terminal antibodies.Permeabilised IFITM2-HA (A) and IFITM3-HA (B) expressing cells were stained with antibodies against the C-terminal HA-tag (green [Alexa-488]) and the NTD, using the anti-IFITM3-NTD antibody (red [Alexa-647]). Images are single optical sections (0.25 µm thick) through the cells. Scale bars represent 15 µm. See also Table 2 and 3 for image analysis.

Mentions: By contrast, on IFITM3-HA expressing cells, a lower level of co-localisation was seen with both NTD antibodies (Fig. 9B and Fig. S6C). Importantly, clear red punctae were visible, suggesting that in some organelles IFITM3 contains intact NTDs but lacks the CTD HA-tag. This conclusion is supported by the quantification of multiple images that demonstrate a lower Mander's M1 and M2, compared to IFITM1-HA, and show an excess of red pixels (55% [±1.4%]) for IFITM3-HA expressing cells (Table 2, 3 and Table S1).


A membrane topology model for human interferon inducible transmembrane protein 1.

Weston S, Czieso S, White IJ, Smith SE, Kellam P, Marsh M - PLoS ONE (2014)

Co-staining with N- and C-terminal antibodies.Permeabilised IFITM2-HA (A) and IFITM3-HA (B) expressing cells were stained with antibodies against the C-terminal HA-tag (green [Alexa-488]) and the NTD, using the anti-IFITM3-NTD antibody (red [Alexa-647]). Images are single optical sections (0.25 µm thick) through the cells. Scale bars represent 15 µm. See also Table 2 and 3 for image analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126714&req=5

pone-0104341-g009: Co-staining with N- and C-terminal antibodies.Permeabilised IFITM2-HA (A) and IFITM3-HA (B) expressing cells were stained with antibodies against the C-terminal HA-tag (green [Alexa-488]) and the NTD, using the anti-IFITM3-NTD antibody (red [Alexa-647]). Images are single optical sections (0.25 µm thick) through the cells. Scale bars represent 15 µm. See also Table 2 and 3 for image analysis.
Mentions: By contrast, on IFITM3-HA expressing cells, a lower level of co-localisation was seen with both NTD antibodies (Fig. 9B and Fig. S6C). Importantly, clear red punctae were visible, suggesting that in some organelles IFITM3 contains intact NTDs but lacks the CTD HA-tag. This conclusion is supported by the quantification of multiple images that demonstrate a lower Mander's M1 and M2, compared to IFITM1-HA, and show an excess of red pixels (55% [±1.4%]) for IFITM3-HA expressing cells (Table 2, 3 and Table S1).

Bottom Line: Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular.Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins.This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.

ABSTRACT
InterFeron Inducible TransMembrane proteins 1-3 (IFITM1, IFITM2 and IFITM3) are a family of proteins capable of inhibiting the cellular entry of numerous human and animal viruses. IFITM1-3 are unique amongst the currently described viral restriction factors in their apparent ability to block viral entry. This restrictive property is dependant on the localisation of the proteins to plasma and endosomal membranes, which constitute the main portals of viral entry into cells. The topology of the IFITM proteins within cell membranes is an unresolved aspect of their biology. Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular. Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins. This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

Show MeSH
Related in: MedlinePlus