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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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Related in: MedlinePlus

Trypsin cleavage of HA-tagged IFITM1.A) Predicted trypsin cleavage sites in hu IFITM1 CTD (Model 3, Fig. 1). B) IFITM1-HA cells were treated with exogenous trypsin for 5 to 30 mins at 37°C. The trypsin was inactivated with soybean trypsin inhibitor, the cells were lysed and the cellular proteins separated by SDS-PAGE. After transfer, proteins were identified with anti-IFITM1-NTD (i) and anti-HA (ii) antibodies. VDAC was used as a loading control (iii). Control samples were untreated (UN), or treated with SBTI-inactivated trypsin (IN). In the overlay image, red represents anti-IFITM1-NTD labelling and green represents anti-HA labelling (iv).
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pone-0104341-g005: Trypsin cleavage of HA-tagged IFITM1.A) Predicted trypsin cleavage sites in hu IFITM1 CTD (Model 3, Fig. 1). B) IFITM1-HA cells were treated with exogenous trypsin for 5 to 30 mins at 37°C. The trypsin was inactivated with soybean trypsin inhibitor, the cells were lysed and the cellular proteins separated by SDS-PAGE. After transfer, proteins were identified with anti-IFITM1-NTD (i) and anti-HA (ii) antibodies. VDAC was used as a loading control (iii). Control samples were untreated (UN), or treated with SBTI-inactivated trypsin (IN). In the overlay image, red represents anti-IFITM1-NTD labelling and green represents anti-HA labelling (iv).

Mentions: Immunofluorescence microscopy (Fig. 2 and 4) suggested that the IFITM1 C-terminal HA-tag resides on the extracellular face of the plasma membrane, while the NTD is cytoplasmic. To further investigate this topology, we used protease cleavage assays. Since IFITM1-HA is primarily localised to the plasma membrane we hypothesised that the CTD, and HA-tag, might be accessible to digestion by extracellular proteases. Following analysis of the human IFITM1 CTD sequence with the ExPASy ‘PeptideCutter’ tool (http://web.expasy.org/peptide_cutter/) we chose to use trypsin, which was predicted to cleave at two positions close to the C-terminus of hu IFITM1 and thus expected to release the HA-tag (Fig. 5A).


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)

Trypsin cleavage of HA-tagged IFITM1.A) Predicted trypsin cleavage sites in hu IFITM1 CTD (Model 3, Fig. 1). B) IFITM1-HA cells were treated with exogenous trypsin for 5 to 30 mins at 37°C. The trypsin was inactivated with soybean trypsin inhibitor, the cells were lysed and the cellular proteins separated by SDS-PAGE. After transfer, proteins were identified with anti-IFITM1-NTD (i) and anti-HA (ii) antibodies. VDAC was used as a loading control (iii). Control samples were untreated (UN), or treated with SBTI-inactivated trypsin (IN). In the overlay image, red represents anti-IFITM1-NTD labelling and green represents anti-HA labelling (iv).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104341-g005: Trypsin cleavage of HA-tagged IFITM1.A) Predicted trypsin cleavage sites in hu IFITM1 CTD (Model 3, Fig. 1). B) IFITM1-HA cells were treated with exogenous trypsin for 5 to 30 mins at 37°C. The trypsin was inactivated with soybean trypsin inhibitor, the cells were lysed and the cellular proteins separated by SDS-PAGE. After transfer, proteins were identified with anti-IFITM1-NTD (i) and anti-HA (ii) antibodies. VDAC was used as a loading control (iii). Control samples were untreated (UN), or treated with SBTI-inactivated trypsin (IN). In the overlay image, red represents anti-IFITM1-NTD labelling and green represents anti-HA labelling (iv).
Mentions: Immunofluorescence microscopy (Fig. 2 and 4) suggested that the IFITM1 C-terminal HA-tag resides on the extracellular face of the plasma membrane, while the NTD is cytoplasmic. To further investigate this topology, we used protease cleavage assays. Since IFITM1-HA is primarily localised to the plasma membrane we hypothesised that the CTD, and HA-tag, might be accessible to digestion by extracellular proteases. Following analysis of the human IFITM1 CTD sequence with the ExPASy ‘PeptideCutter’ tool (http://web.expasy.org/peptide_cutter/) we chose to use trypsin, which was predicted to cleave at two positions close to the C-terminus of hu IFITM1 and thus expected to release the HA-tag (Fig. 5A).

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