GRIF-1–kinesin-1 interactions: a confocal microscopy study

GRIF-1 [GABA_A (γ-aminobutyric acid_A) receptor interacting factor-1] is a member of a coiled-coil family of proteins thought to function as adaptors in the anterograde trafficking of organelles utilizing the kinesin-1 motor proteins to synapses. To study in more detail the molecular interaction between GRIF-1 and the kinesin-1 family member KIF5C, fluorescent yellow- and fluorescent cyan-tagged GRIF-1, KIF5C, the KIF5C MD (motor domain) and the KIF5C NMD (non-motor domain) fusion proteins were generated. Each was characterized with respect to size and ability to co-associate by immunoprecipitation following expression in HEK-293 (human embryonic kidney 293) cells. Further, their distribution in transfected HEK-293 and transformed African green monkey kidney (COS-7) cells was analysed by confocal microscopy. The fluorescent GRIF-1 and KIF5C fusion proteins were all found to behave as wild-type. Double GRIF-1/KIF5C transfectants revealed co-localization. The GRIF-1/KIF5C and GRIF-1/KIF5C NMD double transfectants showed different subcellular distributions compared with single GRIF-1, KIF5C or KIF5C NMD transfections. These studies confirm the association between GRIF-1 and kinesin-1 NMDs. Fluorescence resonance energy transfer studies are ongoing to characterize this interaction in more detail.