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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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Biotin labelling and pulldown of untagged IFITM1.Untagged, wild type IFITM1 or IFITM1-Vstop expression plasmids were transfected into HEK293T cells. After two days the cells were labelled with cell impermeable Sulfo-NHS-SS-Biotin prior to incubation with NeutrAvidin agarose beads. A) Diagram to show the exposed CTD of IFITM1, with the targeted K122, or IFITM1-Vstop. B) Western blots probed with anti-IFITM1-NTD; WCL – whole cell lysates, UB – material that remained unbound by NeutrAvidin, PD1 and PD2 – two rounds of elution of protein from the NeutrAvidin beads. Gel i shows samples from cells labelled with biotin, gel ii shows unlabelled samples and gel iii shows samples from mock transfected HEK293T cells that were treated with Sulfo-NHS-SS-Biotin. NB. The elution step detached some NeutrAvidin monomers from the beads. These run at approximately 14 KDa and are seen as background bands in the western blots (labelled ‘NeutrAvidin’). Calreticulin was used as a loading control and negative control for pulldown specificity. C) Western blot comparing the wild type IFITM1 (M1) with IFITM1-Vstop (Vstop), along with mock transfected HEK293T cells. D) Western blots probed with anti-IFITM1-NTD for whole cell lysates (i) material that remained unbound to NeutrAvidin (ii) and protein eluted from the NeutrAvidin beads (iii). As previously, NeutrAvidin monomers were eluted, and have the same molecular weight at IFITM1-Vstop. This can be clearly seen in the pulldown blot due to the presence of a band in the unlabelled lane.
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pone-0104341-g006: Biotin labelling and pulldown of untagged IFITM1.Untagged, wild type IFITM1 or IFITM1-Vstop expression plasmids were transfected into HEK293T cells. After two days the cells were labelled with cell impermeable Sulfo-NHS-SS-Biotin prior to incubation with NeutrAvidin agarose beads. A) Diagram to show the exposed CTD of IFITM1, with the targeted K122, or IFITM1-Vstop. B) Western blots probed with anti-IFITM1-NTD; WCL – whole cell lysates, UB – material that remained unbound by NeutrAvidin, PD1 and PD2 – two rounds of elution of protein from the NeutrAvidin beads. Gel i shows samples from cells labelled with biotin, gel ii shows unlabelled samples and gel iii shows samples from mock transfected HEK293T cells that were treated with Sulfo-NHS-SS-Biotin. NB. The elution step detached some NeutrAvidin monomers from the beads. These run at approximately 14 KDa and are seen as background bands in the western blots (labelled ‘NeutrAvidin’). Calreticulin was used as a loading control and negative control for pulldown specificity. C) Western blot comparing the wild type IFITM1 (M1) with IFITM1-Vstop (Vstop), along with mock transfected HEK293T cells. D) Western blots probed with anti-IFITM1-NTD for whole cell lysates (i) material that remained unbound to NeutrAvidin (ii) and protein eluted from the NeutrAvidin beads (iii). As previously, NeutrAvidin monomers were eluted, and have the same molecular weight at IFITM1-Vstop. This can be clearly seen in the pulldown blot due to the presence of a band in the unlabelled lane.

Mentions: To exclude the possibility that C-terminal tagging influences the topology of IFITM1, we made use of a lysine residue (K122) present in the CTD that, if the observed topology is correct, will be accessible to labelling with NHS-biotin (Fig. 6A). To investigate this hypothesis, HEK293T cells were transfected with an untagged IFITM1 expression plasmid and incubated with cell impermeable Sulfo-NHS-SS-Biotin for 45 min at 37°C. While HEK293T cells appear to have a low level of IFITM1 mRNA (Fig. S4), no protein was detected by western blot of mock transfected cells (Fig. 6Biii).


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)

Biotin labelling and pulldown of untagged IFITM1.Untagged, wild type IFITM1 or IFITM1-Vstop expression plasmids were transfected into HEK293T cells. After two days the cells were labelled with cell impermeable Sulfo-NHS-SS-Biotin prior to incubation with NeutrAvidin agarose beads. A) Diagram to show the exposed CTD of IFITM1, with the targeted K122, or IFITM1-Vstop. B) Western blots probed with anti-IFITM1-NTD; WCL – whole cell lysates, UB – material that remained unbound by NeutrAvidin, PD1 and PD2 – two rounds of elution of protein from the NeutrAvidin beads. Gel i shows samples from cells labelled with biotin, gel ii shows unlabelled samples and gel iii shows samples from mock transfected HEK293T cells that were treated with Sulfo-NHS-SS-Biotin. NB. The elution step detached some NeutrAvidin monomers from the beads. These run at approximately 14 KDa and are seen as background bands in the western blots (labelled ‘NeutrAvidin’). Calreticulin was used as a loading control and negative control for pulldown specificity. C) Western blot comparing the wild type IFITM1 (M1) with IFITM1-Vstop (Vstop), along with mock transfected HEK293T cells. D) Western blots probed with anti-IFITM1-NTD for whole cell lysates (i) material that remained unbound to NeutrAvidin (ii) and protein eluted from the NeutrAvidin beads (iii). As previously, NeutrAvidin monomers were eluted, and have the same molecular weight at IFITM1-Vstop. This can be clearly seen in the pulldown blot due to the presence of a band in the unlabelled lane.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104341-g006: Biotin labelling and pulldown of untagged IFITM1.Untagged, wild type IFITM1 or IFITM1-Vstop expression plasmids were transfected into HEK293T cells. After two days the cells were labelled with cell impermeable Sulfo-NHS-SS-Biotin prior to incubation with NeutrAvidin agarose beads. A) Diagram to show the exposed CTD of IFITM1, with the targeted K122, or IFITM1-Vstop. B) Western blots probed with anti-IFITM1-NTD; WCL – whole cell lysates, UB – material that remained unbound by NeutrAvidin, PD1 and PD2 – two rounds of elution of protein from the NeutrAvidin beads. Gel i shows samples from cells labelled with biotin, gel ii shows unlabelled samples and gel iii shows samples from mock transfected HEK293T cells that were treated with Sulfo-NHS-SS-Biotin. NB. The elution step detached some NeutrAvidin monomers from the beads. These run at approximately 14 KDa and are seen as background bands in the western blots (labelled ‘NeutrAvidin’). Calreticulin was used as a loading control and negative control for pulldown specificity. C) Western blot comparing the wild type IFITM1 (M1) with IFITM1-Vstop (Vstop), along with mock transfected HEK293T cells. D) Western blots probed with anti-IFITM1-NTD for whole cell lysates (i) material that remained unbound to NeutrAvidin (ii) and protein eluted from the NeutrAvidin beads (iii). As previously, NeutrAvidin monomers were eluted, and have the same molecular weight at IFITM1-Vstop. This can be clearly seen in the pulldown blot due to the presence of a band in the unlabelled lane.
Mentions: To exclude the possibility that C-terminal tagging influences the topology of IFITM1, we made use of a lysine residue (K122) present in the CTD that, if the observed topology is correct, will be accessible to labelling with NHS-biotin (Fig. 6A). To investigate this hypothesis, HEK293T cells were transfected with an untagged IFITM1 expression plasmid and incubated with cell impermeable Sulfo-NHS-SS-Biotin for 45 min at 37°C. While HEK293T cells appear to have a low level of IFITM1 mRNA (Fig. S4), no protein was detected by western blot of mock transfected cells (Fig. 6Biii).

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