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The light-dependent subcellular translocation of rod alpha-transducin (GNAT-1, or rod Talpha) has been well documented. In dark-adapted animals, rod Talpha (rTalpha) is predominantly located in the rod outer segment (ROS) and translocates into the rod inner segment (RIS) upon exposure to the light. Neither the molecular participants nor the mechanism(s) involved in this protein trafficking are known. We hypothesized that other proteins must interact with rTalpha to affect the translocations. Using the MBP-rTalpha fusion pulldown assay, the yeast two-hybrid assay and the co-immunoprecipitation assay, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rTalpha as interacting proteins. Immunoprecipitation also showed beta-actin associates with rTalpha in the dark but not in the light. To further investigate the involvement of GAPDH in light-induced rod Talpha translocation, GAPDH mRNA was knocked down in vivo by transient expression of siRNAs in rat photoreceptor cells. Under completely dark- and light-adapted conditions, the translocation of rTalpha was not significantly different within the 'GAPDH knock-down photoreceptor cells' compared to the non-transfected control cells. However, under partial dark-adaptation, rTalpha translocated more slowly in the 'GAPDH knock-down cells' supporting the conclusion that GAPDH is involved in rTalpha translocation from the RIS to the ROS during dark adaptation.