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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 IFITM NTD antibodies.Two commercially available antibodies targeting either the IFITM1-NTD or the IFITM3-NTD were screened by western blot to assess specificity using HA-tagged IFITM1-3 (M1, M2 and M3) cell lines along with control A549 cells. Proteins were also identified using the HA epitope. Blots were imaged on a Li-COR Odyssey system that uses far-red fluorophore conjugated secondary antibodies. In the overlay image, red represents anti-IFITM-NTD labelling and green represents anti-HA labelling. A) Anti-IFITM1-NTD detects IFITM1 and shows cross-reactivity with IFITM3. B) Anti-IFITM3-NTD detects IFITM3 and has cross-reactivity with IFITM2. VDAC was used as a loading control.
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pone-0104341-g003: Analysis of IFITM NTD antibodies.Two commercially available antibodies targeting either the IFITM1-NTD or the IFITM3-NTD were screened by western blot to assess specificity using HA-tagged IFITM1-3 (M1, M2 and M3) cell lines along with control A549 cells. Proteins were also identified using the HA epitope. Blots were imaged on a Li-COR Odyssey system that uses far-red fluorophore conjugated secondary antibodies. In the overlay image, red represents anti-IFITM-NTD labelling and green represents anti-HA labelling. A) Anti-IFITM1-NTD detects IFITM1 and shows cross-reactivity with IFITM3. B) Anti-IFITM3-NTD detects IFITM3 and has cross-reactivity with IFITM2. VDAC was used as a loading control.

Mentions: To determine the specificity of the NTD antibodies, both were tested on samples from the HA-tagged IFITM1-3 expressing A549 cells by western blot analysis. Anti-IFITM1-NTD detected IFITM1-HA and IFITM3-HA, but not IFITM2-HA (Fig. 3A). Anti-IFITM3-NTD detected both IFITM3-HA and IFITM2-HA, but not IFITM1-HA (Fig. 3B). No IFITM protein was detected in control A549 cells with either antibody by western blot. Multiple bands in the range of 12–17 kDa were seen with the NTD antibodies, but not the anti-HA, indicating possible post-translational modification of the proteins (see below).


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 IFITM NTD antibodies.Two commercially available antibodies targeting either the IFITM1-NTD or the IFITM3-NTD were screened by western blot to assess specificity using HA-tagged IFITM1-3 (M1, M2 and M3) cell lines along with control A549 cells. Proteins were also identified using the HA epitope. Blots were imaged on a Li-COR Odyssey system that uses far-red fluorophore conjugated secondary antibodies. In the overlay image, red represents anti-IFITM-NTD labelling and green represents anti-HA labelling. A) Anti-IFITM1-NTD detects IFITM1 and shows cross-reactivity with IFITM3. B) Anti-IFITM3-NTD detects IFITM3 and has cross-reactivity with IFITM2. VDAC was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104341-g003: Analysis of IFITM NTD antibodies.Two commercially available antibodies targeting either the IFITM1-NTD or the IFITM3-NTD were screened by western blot to assess specificity using HA-tagged IFITM1-3 (M1, M2 and M3) cell lines along with control A549 cells. Proteins were also identified using the HA epitope. Blots were imaged on a Li-COR Odyssey system that uses far-red fluorophore conjugated secondary antibodies. In the overlay image, red represents anti-IFITM-NTD labelling and green represents anti-HA labelling. A) Anti-IFITM1-NTD detects IFITM1 and shows cross-reactivity with IFITM3. B) Anti-IFITM3-NTD detects IFITM3 and has cross-reactivity with IFITM2. VDAC was used as a loading control.
Mentions: To determine the specificity of the NTD antibodies, both were tested on samples from the HA-tagged IFITM1-3 expressing A549 cells by western blot analysis. Anti-IFITM1-NTD detected IFITM1-HA and IFITM3-HA, but not IFITM2-HA (Fig. 3A). Anti-IFITM3-NTD detected both IFITM3-HA and IFITM2-HA, but not IFITM1-HA (Fig. 3B). No IFITM protein was detected in control A549 cells with either antibody by western blot. Multiple bands in the range of 12–17 kDa were seen with the NTD antibodies, but not the anti-HA, indicating possible post-translational modification of the proteins (see below).

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