Fig. 2

LITAF is anchored to membranes via hydrophobic residues contained within the LITAF domain. a Amino acid sequences of the hydrophobic regions present in the protein constructs used to determine membrane insertion. Residues are numbered according to the full length human LITAF sequence. Hydrophobic residues are coloured red, while acidic residues are coloured blue. Residues retained in all three constructs are indicated by asterisks below the sequence. The grey box denotes the serine–glycine linker added in place of the predicted helix in the Δ114–139 construct. b The HA-tagged LITAF constructs were transiently expressed in HeLa cells and the localisation determined by immunofluorescence microscopy. The endosomal targeting of endogenously expressed VPS26 is shown in the left panels for comparison. The middle panels show that the endosomal membrane targeting of LITAF is dependent on key hydrophobic residues within the hydrophobic region. Scale bar denotes 20 μm. c Membrane fractionation from HeLa cells expressing endogenous LITAF and transiently expressed HA-tagged LITAF constructs. While both endogenous and HA-tagged wild type LITAF is found in the membrane pellets (P), the LITAF constructs harbouring hydrophilic mutations (N-helix) and a deletion in the vicinity of the hydrophobic region (Δ114–139) are found in the soluble fraction (S). Calnexin and tubulin were used as integral membrane and soluble protein controls, respectively. d Wild type LITAF displays the characteristics of an integral membrane protein as determined by the extraction of the endogenous protein from HeLa cells using TX114. The integral membrane protein, Calnexin, and VPS26, which associates with, but does not insert into membranes, were used as controls