FIGURE 1.

The GXXXG motif mediates self-assembly of the TMD but is insufficient to drive VAP-B oligomerization. A, the oligomerization of Myc-tagged wild-type and VAP-B mutants was assessed following cross-linking (CL) of HEK293 cells expressing the indicated VAP-B proteins, using Western blotting with anti-Myc antibody. Arrows mark the positions of the monomer, dimer, and tetramer. The following VAP-B proteins were examined: wild-type (WT), the G235I (I) mutant, the G235/239I (II) double mutant, the FFAT-binding mutant K87/M89D, the K87D/M89D/G235I; K87/M89D/(I) triple mutant, and the K87D/M89D/G235I/G239I; K87/M89D/(II) mutant. B, the indicated HA-tagged VAP-B proteins were expressed in HEK293 cells either alone or together with the indicated Myc-tagged VAP-B. The interaction between the VAP-B proteins was determined by immunoprecipitation with anti-Myc antibody followed by immunoblotting with anti-HA antibody. C, HEK293 cells expressing the TMD of VAP-B (WT) or the II mutant fused to monomeric YFP (A206K) were cross-linked by formaldehyde, and their oligomerization was assessed by Western blotting using anti-GFP antibody before and after cross-linker cleavage, CL and CL-Rev., respectively. Arrows mark the positions of the monomer and dimer. The weak band that appears above the dimer might represent a complex between YFP-TMD and endogenous VAP-B (or -A). Positions of prestained molecular mass markers, in kDa, are indicated on the left.