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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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Analysis of the cellular distribution of the human IFITM proteins using anti-N-terminal domain antibodies.Immunofluorescence of A549 cells expressing HA-tagged IFITM1, IFITM2 and IFITM3 (or control A549 cells) using anti-IFITM1-NTD and anti-IFITM3 NTD antibodies. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA show distinct distributions in permeabilised cells. E and F) Anti-IFITM3-NTD detects low levels of endogenous protein in control A549 and IFITM1-HA cell lines. G and H) Anti-IFITM3-NTD detects IFITM2-HA and IFITM3-HA in permeabilised cells. I-L) No specific staining was seen on intact cells labelled with anti-IFITM1-NTD antibody. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells on a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.
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pone-0104341-g004: Analysis of the cellular distribution of the human IFITM proteins using anti-N-terminal domain antibodies.Immunofluorescence of A549 cells expressing HA-tagged IFITM1, IFITM2 and IFITM3 (or control A549 cells) using anti-IFITM1-NTD and anti-IFITM3 NTD antibodies. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA show distinct distributions in permeabilised cells. E and F) Anti-IFITM3-NTD detects low levels of endogenous protein in control A549 and IFITM1-HA cell lines. G and H) Anti-IFITM3-NTD detects IFITM2-HA and IFITM3-HA in permeabilised cells. I-L) No specific staining was seen on intact cells labelled with anti-IFITM1-NTD antibody. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells on a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.

Mentions: When intact IFITM1, 2 or 3 cells were labelled with anti-IFITM1-NTD antibody, no staining was seen, suggesting intracellular NTDs (Fig. 4J, K and L). Following detergent treatment, all three IFITM expressing lines were labelled (Fig. 4B, C and D), though a weaker signal was seen for IFITM2, as expected from the western blot (Fig. 3A). By immunofluorescence, the anti-IFITM3-NTD antibody gave a low signal in the untransfected A549 control and IFITM1-HA cells (Fig. 4E and D), consistent with the low level of endogenous IFITM2 expression detected by qRT-PCR (Fig. S4). However, the antibody clearly detected IFITM2-HA and IFITM3-HA (Fig. 4G and H). The observed cellular distributions, using the anti-NTD antibodies, agree with those seen using anti-HA antibodies (Fig. 2), and the inaccessibility of the IFITM1-NTD to labelling on intact cells indicates the domain is on the cytoplasmic side of the plasma membrane.


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)

Analysis of the cellular distribution of the human IFITM proteins using anti-N-terminal domain antibodies.Immunofluorescence of A549 cells expressing HA-tagged IFITM1, IFITM2 and IFITM3 (or control A549 cells) using anti-IFITM1-NTD and anti-IFITM3 NTD antibodies. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA show distinct distributions in permeabilised cells. E and F) Anti-IFITM3-NTD detects low levels of endogenous protein in control A549 and IFITM1-HA cell lines. G and H) Anti-IFITM3-NTD detects IFITM2-HA and IFITM3-HA in permeabilised cells. I-L) No specific staining was seen on intact cells labelled with anti-IFITM1-NTD antibody. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells on a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104341-g004: Analysis of the cellular distribution of the human IFITM proteins using anti-N-terminal domain antibodies.Immunofluorescence of A549 cells expressing HA-tagged IFITM1, IFITM2 and IFITM3 (or control A549 cells) using anti-IFITM1-NTD and anti-IFITM3 NTD antibodies. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA show distinct distributions in permeabilised cells. E and F) Anti-IFITM3-NTD detects low levels of endogenous protein in control A549 and IFITM1-HA cell lines. G and H) Anti-IFITM3-NTD detects IFITM2-HA and IFITM3-HA in permeabilised cells. I-L) No specific staining was seen on intact cells labelled with anti-IFITM1-NTD antibody. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells on a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.
Mentions: When intact IFITM1, 2 or 3 cells were labelled with anti-IFITM1-NTD antibody, no staining was seen, suggesting intracellular NTDs (Fig. 4J, K and L). Following detergent treatment, all three IFITM expressing lines were labelled (Fig. 4B, C and D), though a weaker signal was seen for IFITM2, as expected from the western blot (Fig. 3A). By immunofluorescence, the anti-IFITM3-NTD antibody gave a low signal in the untransfected A549 control and IFITM1-HA cells (Fig. 4E and D), consistent with the low level of endogenous IFITM2 expression detected by qRT-PCR (Fig. S4). However, the antibody clearly detected IFITM2-HA and IFITM3-HA (Fig. 4G and H). The observed cellular distributions, using the anti-NTD antibodies, agree with those seen using anti-HA antibodies (Fig. 2), and the inaccessibility of the IFITM1-NTD to labelling on intact cells indicates the domain is on the cytoplasmic side of the plasma membrane.

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