Gaf-1, a γ-SNAP-binding protein associated with the mitochondria

The role of alpha/beta -SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma -SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma -SNAP binding proteins and to uncover potentially novel functions for gamma -SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma -SNAP associate factor-1) specifically binds gamma- but not alpha -SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma -SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha -SNAP or NSF coprecipitated with Gaf-1. Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of gamma -SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma -SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells. This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements. These data identify a class of gamma -SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma -SNAP in mitochondrial dynamics.