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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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Immuno-gold labelling of A549 IFITM1-HA cell cryo-sections.Cryo-sections of the IFITM1-HA cells were labelled with anti-HA antibodies and Protein A gold. A) Plasma membrane labelling. B and C) Plasma membrane and multi-vesicular body labelling. D) Plasma membrane and Golgi apparatus labelling. Scale bars represent 200 nm.
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pone-0104341-g007: Immuno-gold labelling of A549 IFITM1-HA cell cryo-sections.Cryo-sections of the IFITM1-HA cells were labelled with anti-HA antibodies and Protein A gold. A) Plasma membrane labelling. B and C) Plasma membrane and multi-vesicular body labelling. D) Plasma membrane and Golgi apparatus labelling. Scale bars represent 200 nm.

Mentions: To analyse hu IFITM1 in more detail, cryo-sections of IFITM1-HA cells were immuno-gold labelled for the HA-tag and examined by electron microscopy. As expected, labelling was seen predominantly at the plasma membrane (Fig. 7A and B). Close examination indicated that the majority of gold particles resided on the extracellular face of the plasma membrane, consistent with the notion that the hu IFITM1 CTD is located extracellularly. Labelling was also seen in the Golgi apparatus and in multi-vesicular bodies (Fig. 7C and D). These intracellular pools presumably indicate IFITM1 trafficking in the biosynthetic and endocytic pathways. This material would be inaccessible to extracellular proteases and labelling reagents, explaining the failure to completely digest IFITM1-HA with trypsin (Fig. 5B) and to fully label IFITM1 with biotin (Fig. 6B).


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)

Immuno-gold labelling of A549 IFITM1-HA cell cryo-sections.Cryo-sections of the IFITM1-HA cells were labelled with anti-HA antibodies and Protein A gold. A) Plasma membrane labelling. B and C) Plasma membrane and multi-vesicular body labelling. D) Plasma membrane and Golgi apparatus labelling. Scale bars represent 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104341-g007: Immuno-gold labelling of A549 IFITM1-HA cell cryo-sections.Cryo-sections of the IFITM1-HA cells were labelled with anti-HA antibodies and Protein A gold. A) Plasma membrane labelling. B and C) Plasma membrane and multi-vesicular body labelling. D) Plasma membrane and Golgi apparatus labelling. Scale bars represent 200 nm.
Mentions: To analyse hu IFITM1 in more detail, cryo-sections of IFITM1-HA cells were immuno-gold labelled for the HA-tag and examined by electron microscopy. As expected, labelling was seen predominantly at the plasma membrane (Fig. 7A and B). Close examination indicated that the majority of gold particles resided on the extracellular face of the plasma membrane, consistent with the notion that the hu IFITM1 CTD is located extracellularly. Labelling was also seen in the Golgi apparatus and in multi-vesicular bodies (Fig. 7C and D). These intracellular pools presumably indicate IFITM1 trafficking in the biosynthetic and endocytic pathways. This material would be inaccessible to extracellular proteases and labelling reagents, explaining the failure to completely digest IFITM1-HA with trypsin (Fig. 5B) and to fully label IFITM1 with biotin (Fig. 6B).

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