Structural basis of FFAT motif-mediated ER targeting

The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain.; This thesis describes the first biophysical characterization of a VAP protein. VAP proteins dimerize in solution via a conserved coiled-coil domain, and the VAP MSP homology domain is structurally similar to, but functionally distinct from MSP Crystal structures of the rat VAP-A MSP homology domain were solved alone and in complexes with FFAT motifs. The VAP-FFAT co-crystal structures were used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant was shown to block the function of the VAP homologue Scs2p in yeast. Furthermore, overexpression of the FFAT binding-defective VAP mutant in COS7 cells dramatically altered ER morphology. Finally, a protease protection assay and preliminary experiments in COS7 cells suggest that VAP may have a function that requires polymerization.; In summary, the data presented establish the structural basis of FFAT mediated ER targeting and suggest that FFAT targeted proteins play an important role in determining ER morphology.