Figure 2

A FFAT-like motif mediates FAF1 interaction with VAPB. (A) The MSP domain of VAPB mediates its interaction with FAF1. Top panel: Schematic representation of the Flag-VAPB constructs, full length (1 to 243) or truncated, C-terminal half (125 to 243) and the MSP domain N-terminal half (1 to 125), immunoprecipitated from U2OS cells as shown in the bottom panel. Immunoblots of inputs (left) and immunoprecipitates (right) show that the MSP of VAPB is sufficient for the interaction with FAF1/p97. A highly conserved FFAT-like motif in FAF1 mediates its interaction with VAPB. (B) Top panel: Schematic representation of human FAF1 highlighting its various domains. Bottom panel: Wild-type or mutant variants of Flag-FAF1 were immunoprecipitated from U2OS cells. The indicated proteins were detected using specific antibodies in the inputs (left) and immunoprecipitates (right). UBA deletion caused a dramatic reduction in ubiquitinated protein binding to FAF1 whereas a point mutation in the UBX domain (P620G) abolished p97 binding. Deletion of the residues 293 to 301 was the only truncation that prevented VAPB binding to FAF1. (C) Alignment of FAF1 sequence from various species showing that the FFAT-like motif is highly conserved. (D) As in (B), showing that either a triple mutation D295A/F296A/E297A or single F296A mutation in FAF1 abolished VAPB binding. IP, immunoprecipitate; WT, wild type.