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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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Cellular distribution of the human IFITM proteins.C-terminal domain HA-tagged IFITM1, IFITM2, IFITM3 and control A549 cell lines were stained intact, or following permeabilisation, with an anti-HA antibody and a secondary anti-rat Alexa-488 antibody. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA have distinct cellular distributions in permeabilised cells. E) Intact A549 cells (no detergent treatment) show no specific staining. F) Intact IFITM1-HA cells show positive cell surface HA staining. G) Intact IFITM2-HA cells show no detectable HA staining. H) Although the majority of intact IFITM3-HA cells show no anti-HA labelling, a minority (<1%) show low-level positive staining. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells using 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-g002: Cellular distribution of the human IFITM proteins.C-terminal domain HA-tagged IFITM1, IFITM2, IFITM3 and control A549 cell lines were stained intact, or following permeabilisation, with an anti-HA antibody and a secondary anti-rat Alexa-488 antibody. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA have distinct cellular distributions in permeabilised cells. E) Intact A549 cells (no detergent treatment) show no specific staining. F) Intact IFITM1-HA cells show positive cell surface HA staining. G) Intact IFITM2-HA cells show no detectable HA staining. H) Although the majority of intact IFITM3-HA cells show no anti-HA labelling, a minority (<1%) show low-level positive staining. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells using a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.

Mentions: To investigate the cellular distributions of human (hu) IFITM1, 2 and 3 we used C-terminally HA-tagged proteins, stably expressed in A549 cells, and immunofluorescence microscopy. When cells were permeabilised and labelled with anti-HA antibodies, all three IFITM proteins were detected, and each had a different cellular distribution. IFITM1-HA was seen primarily at the plasma membrane (Fig. 2B). By contrast, IFITM2-HA and IFITM3-HA were seen mostly in intracellular compartments with distinct distributions. IFITM2-HA localised in a tight cluster of punctae close to the nucleus, while IFITM3-HA had a more dispersed, punctate distribution (Fig. 2C and D).


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)

Cellular distribution of the human IFITM proteins.C-terminal domain HA-tagged IFITM1, IFITM2, IFITM3 and control A549 cell lines were stained intact, or following permeabilisation, with an anti-HA antibody and a secondary anti-rat Alexa-488 antibody. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA have distinct cellular distributions in permeabilised cells. E) Intact A549 cells (no detergent treatment) show no specific staining. F) Intact IFITM1-HA cells show positive cell surface HA staining. G) Intact IFITM2-HA cells show no detectable HA staining. H) Although the majority of intact IFITM3-HA cells show no anti-HA labelling, a minority (<1%) show low-level positive staining. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells using 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-g002: Cellular distribution of the human IFITM proteins.C-terminal domain HA-tagged IFITM1, IFITM2, IFITM3 and control A549 cell lines were stained intact, or following permeabilisation, with an anti-HA antibody and a secondary anti-rat Alexa-488 antibody. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA have distinct cellular distributions in permeabilised cells. E) Intact A549 cells (no detergent treatment) show no specific staining. F) Intact IFITM1-HA cells show positive cell surface HA staining. G) Intact IFITM2-HA cells show no detectable HA staining. H) Although the majority of intact IFITM3-HA cells show no anti-HA labelling, a minority (<1%) show low-level positive staining. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells using a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.
Mentions: To investigate the cellular distributions of human (hu) IFITM1, 2 and 3 we used C-terminally HA-tagged proteins, stably expressed in A549 cells, and immunofluorescence microscopy. When cells were permeabilised and labelled with anti-HA antibodies, all three IFITM proteins were detected, and each had a different cellular distribution. IFITM1-HA was seen primarily at the plasma membrane (Fig. 2B). By contrast, IFITM2-HA and IFITM3-HA were seen mostly in intracellular compartments with distinct distributions. IFITM2-HA localised in a tight cluster of punctae close to the nucleus, while IFITM3-HA had a more dispersed, punctate distribution (Fig. 2C and D).

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