Additional file 6: Figure S6. of The topology, structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C

(a) Schematic diagram illustrating the position of the conserved cysteine residues mutated to alanine in the N-term AxxA and C-term AxxA HA-LITAF constructs. (b) HA-LITAF constructs were transiently expressed in HeLa cells and visualised by immunofluorescence confocal microscopy. In contrast to the wild type LITAF, which targeted multiple punctate structures consistent with endosomes (HA-LITAF WT, left panel), the LITAF constructs in which the N-terminal or C-terminal conserved cysteine pairs were mutated to alanine (HA-LITAF N-term AxxA and HA-LITAF C-term AxxA) appeared in large puncta, consistent with them being proteins prone to aggregation. Nuclei were stained with DAPI. Scale bars denote 10 μm. (c) Membrane fractionation from HeLa cells transiently expressing HA-LITAF constructs. A soluble fraction (Soluble) was obtained following high speed centrifugation. The resulting membrane pellet was further incubated with 1% TX100 in order to extract soluble membrane proteins (TX100) from insoluble material (Pellet). Calnexin and tubulin were used for control purposes as examples of integral membrane and soluble proteins, respectively. While HA-LITAF WT was found in the TX100 solubilised membrane fraction similarly to calnexin, HA-LITAF construct harbouring mutations in either the N-terminal or C-terminal conserved cysteine pairs within the LITAF domain were found predominantly in the TX100 insoluble pellet (P), consistent with protein aggregation. Note that longer exposure times were required for the cysteine mutant samples as the expression level of these mutated proteins was consistently lower compared to wild type. (PDF 246 kb)