M3-HA cells that were not incubated with anti-HA antibodies, showed no labelling. IFITM1-HA expressing cells acted as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19663632 a positive control and showed strong cell BIRB796 chemical information surface labelling. 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 showed intracellular, punctate labelling. 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 Human IFITM1 Membrane Topology fraction of A549 cells, the CTDs of both IFITM2-HA and IFITM3-HA proteins are, at least transiently, exposed on the cell surface. Previous results have suggested that IFITM2 and 3 are localised to endosomes. The exposure of CTD HA-tags in the lumen of these organelles may result in their cleavage by endosomal/lysosomal proteases. This hypothesis is supported by western blots that showed low levels of HA-labelling for IFITM2 and 3. Moreover, when detected with anti-IFITM3-NTD, IFITM2-HA appears to have 3 bands in the range of 1217 kDa, Human IFITM1 Membrane Topology with the majority of the protein being of the lowest molecular weight, consistent with this protein having lost its HA-tag. A similar low molecular weight form was also seen for IFITM3-HA. IFITM expressing A549 cells were co-stained with antibodies against the NTD and the HA-tag. As IFITM proteins are relatively short co-staining for the NTD and CTD should give apparent co-localisation. IFITM1-HA expressing cells showed a high degree of overlap between the antiIFITM1-NTD and anti-HA antibodies. The overlap was seen across multiple images as demonstrated by Mander’s correlation coefficients M1 and M2. Furthermore, analysis of the areas of different pixel colours demonstrated that around 70% of pixels were detectable as yellow. By contrast, on IFITM3-HA expressing cells, a lower level of colocalisation was seen with both NTD antibodies. Importantly, clear red punctae were visible, suggesting that in some organelles IFITM3 contains intact NTDs but lacks the CTD HA-tag. This conclusion is supported by the quantification of multiple images that demonstrate a lower Mander’s M1 and M2, compared to IFITM1-HA, and show an excess of red pixels for IFITM3-HA expressing cells. The anti-IFITM1-NTD antibody does not detect IFITM2-HA as well as IFITM1-HA and IFITM3-HA; we therefore used the anti-IFITM3-NTD antibody for a similar investigation 
of IFITM2-HA. When multiple images were quantified, the results for IFITM2-HA and IFITM3-HA expressing cells were largely equivalent, with 49% of pixels being red for IFITM2-HA cells, compared to 46% for IFITM3-HA cells. The observed excess of red pixels for both IFITM2-HA and IFITM3-HA indicates loss of the HA-tag, consistent with the western blots. Overall, the data suggest a topology in which the HA-tagged CTDs of IFITM2 and IFITM3 are exposed to the endosomal/lysosomal system. Discussion The membrane topology of the interferon-induced IFITM proteins has been controversial. Initially it was suggested that both the NTD and CTD are located on the extracellular face o
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