Gaf-1, a gamma-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 n ew, 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 stoichiometri cally associated with gamma -SNAP in cellular extracts. This binding is dis tinct from other SNAP interactions since no alpha -SNAP or NSF coprecipitat ed with Gaf-1. Subcellular fractionation and immunofluorescence analysis sh ow that Gaf-1 is peripherally associated with the outer mitochondrial membr ane. Only a fraction of gamma -SNAP was mitochondrial with the balance bein g 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 mitoc hondria as well as with microtubules but not with other cytoskeletal elemen ts. These data identify a class of gamma -SNAP interactions that is distinc t from other members of the SNAP family and point to a potential role for g amma -SNAP in mitochondrial dynamics.