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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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Antibody feeding of IFITM expressing cells.Live A549-IFITM-HA cells were incubated with 5 µg/ml anti-HA at 37°C to allow endocytosis of bound antibody molecules. Subsequently, the cells were washed, fixed, permeabilised and incubated with an anti-rat Alexa-488 conjugate. A) Control A549 cells show no specific staining. B) In IFITM1-HA cells the majority of labelling is at the plasma membrane. C) IFITM3-HA cells that were not incubated with anti-HA antibody show no labelling. For IFITM2-HA and IFITM3-HA expressing cells (D and E) the majority of cells are not labelled, however in both cases a minority of cells do show punctate labelling indicative of IFITM-mediated internalisation of anti-HA antibodies. In D and E, the boxed region has been enlarged. All images were taken using the same microscope settings and adjusted uniformly. Scale bars represent 15 µm.
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pone-0104341-g008: Antibody feeding of IFITM expressing cells.Live A549-IFITM-HA cells were incubated with 5 µg/ml anti-HA at 37°C to allow endocytosis of bound antibody molecules. Subsequently, the cells were washed, fixed, permeabilised and incubated with an anti-rat Alexa-488 conjugate. A) Control A549 cells show no specific staining. B) In IFITM1-HA cells the majority of labelling is at the plasma membrane. C) IFITM3-HA cells that were not incubated with anti-HA antibody show no labelling. For IFITM2-HA and IFITM3-HA expressing cells (D and E) the majority of cells are not labelled, however in both cases a minority of cells do show punctate labelling indicative of IFITM-mediated internalisation of anti-HA antibodies. In D and E, the boxed region has been enlarged. All images were taken using the same microscope settings and adjusted uniformly. Scale bars represent 15 µm.

Mentions: An antibody-feeding approach was used in which live cells were cultured in medium containing anti-HA antibody for 3 h, prior to washing, fixation and visualisation by immunofluorescence microscopy. As expected, control untransfected A549 cells, incubated with anti-HA antibodies, and IFITM3-HA cells that were not incubated with anti-HA antibodies, showed no labelling (Fig. 8A and C). IFITM1-HA expressing cells acted as a positive control and showed strong cell surface labelling (Fig. 8B). By contrast, most IFITM2-HA and IFITM3-HA cells showed little, if any, labelling. However, approximately 25% of IFITM3-HA cells and 10% of IFITM2-HA cells (calculated from 30 random fields of view at 40X magnification) showed intracellular, punctate labelling (Fig. 8D and E). Since the cells were labelled prior to fixation, this intracellular labelling suggested that, in some cells, the IFITM2-HA and IFITM3-HA proteins are trafficked to the cell surface where an externally exposed HA epitope could bind antibody, prior to internalisation into intracellular organelles. The lack of labelling in the majority of cells, which express the IFITM proteins, indicates the labelling is not due to non-specific fluid phase uptake of antibody. Together, we conclude that, in a fraction of A549 cells, the CTDs of both IFITM2-HA and IFITM3-HA proteins are, at least transiently, exposed on the cell surface.


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)

Antibody feeding of IFITM expressing cells.Live A549-IFITM-HA cells were incubated with 5 µg/ml anti-HA at 37°C to allow endocytosis of bound antibody molecules. Subsequently, the cells were washed, fixed, permeabilised and incubated with an anti-rat Alexa-488 conjugate. A) Control A549 cells show no specific staining. B) In IFITM1-HA cells the majority of labelling is at the plasma membrane. C) IFITM3-HA cells that were not incubated with anti-HA antibody show no labelling. For IFITM2-HA and IFITM3-HA expressing cells (D and E) the majority of cells are not labelled, however in both cases a minority of cells do show punctate labelling indicative of IFITM-mediated internalisation of anti-HA antibodies. In D and E, the boxed region has been enlarged. All images were taken using the same microscope settings and adjusted uniformly. Scale bars represent 15 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104341-g008: Antibody feeding of IFITM expressing cells.Live A549-IFITM-HA cells were incubated with 5 µg/ml anti-HA at 37°C to allow endocytosis of bound antibody molecules. Subsequently, the cells were washed, fixed, permeabilised and incubated with an anti-rat Alexa-488 conjugate. A) Control A549 cells show no specific staining. B) In IFITM1-HA cells the majority of labelling is at the plasma membrane. C) IFITM3-HA cells that were not incubated with anti-HA antibody show no labelling. For IFITM2-HA and IFITM3-HA expressing cells (D and E) the majority of cells are not labelled, however in both cases a minority of cells do show punctate labelling indicative of IFITM-mediated internalisation of anti-HA antibodies. In D and E, the boxed region has been enlarged. All images were taken using the same microscope settings and adjusted uniformly. Scale bars represent 15 µm.
Mentions: An antibody-feeding approach was used in which live cells were cultured in medium containing anti-HA antibody for 3 h, prior to washing, fixation and visualisation by immunofluorescence microscopy. As expected, control untransfected A549 cells, incubated with anti-HA antibodies, and IFITM3-HA cells that were not incubated with anti-HA antibodies, showed no labelling (Fig. 8A and C). IFITM1-HA expressing cells acted as a positive control and showed strong cell surface labelling (Fig. 8B). By contrast, most IFITM2-HA and IFITM3-HA cells showed little, if any, labelling. However, approximately 25% of IFITM3-HA cells and 10% of IFITM2-HA cells (calculated from 30 random fields of view at 40X magnification) showed intracellular, punctate labelling (Fig. 8D and E). Since the cells were labelled prior to fixation, this intracellular labelling suggested that, in some cells, the IFITM2-HA and IFITM3-HA proteins are trafficked to the cell surface where an externally exposed HA epitope could bind antibody, prior to internalisation into intracellular organelles. The lack of labelling in the majority of cells, which express the IFITM proteins, indicates the labelling is not due to non-specific fluid phase uptake of antibody. Together, we conclude that, in a fraction of A549 cells, the CTDs of both IFITM2-HA and IFITM3-HA proteins are, at least transiently, exposed on the cell surface.

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