Cotransport of glyceraldehyde-3-phosphate dehydrogenase and actin in axonsof chicken motoneurons

1. To study proteins transported with actin in axons, we pulse-labeled moto neurons in the chicken sciatic nerve with [S-35]methionine and, 1-20 days l atter, isolated actin and its binding proteins by affinity chromatography o f Triton soluble nerve extracts on DNase I-Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific acti vity of the radioactively labeled protein spots was estimated by fluorograp hy. 2. In addition to actin, which binds specifically to DNase I, a small numbe r of other proteins were labeled, including established actin monomer bindi ng proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecul ar mass, pI, amino acid composition, and immunostaining, the unrecognized p rotein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate d ehydrogenase (GAPDH). 3. The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin ) and indirect immunomagnetic separation with Dynabeads covalently linked t o sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresi s and immunoblotting showed that actin and GAPDH were the main proteins iso lated by these methods. 4. Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3-5 mm/day, which corresponds to slow component b of axonal transport. These proteins w ere not associated with rapidly transported proteins that accumulated proxi mal to a ligation 7 cm from the spinal cord 9 hr after injection of radioac tivity. 5. Our results indicate that GAPDH and actin are transported as a complex i n axons and raise the possibility that GAPDH could act as a chaperone for m onomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in tr anslocating and anchoring GAPDH to specialized sites in axons and nerve ter minals that require a source of ATP by glycolysis.