Characterization of posttranslational processing of the mammalian high-molecular-weight neurofilament protein in vivo.
Sensory neurons in the dorsal root ganglion (DRG) were used in an in vivo pulse-chase labeling paradigm to examine the time course and nature of posttranslational processing of neurofilament (NF) proteins. Ganglia of adult rats were labeled with 35S-methionine and harvested 1-72 hr later. Samples containing the cell bodies and short initial axonal segments of DRG neurons were analyzed by 1- and 2-dimensional PAGE/fluorography. For comparison, axonally transported NF proteins (200, 145, and 68 kDa) were harvested from the sciatic nerve 21 d after labeling the fifth lumbar (L5) DRG. Analysis of the pulse-chase experiments revealed that the mature 200 kDa protein (NF200) was not identifiable in gels of DRG samples until 24-48 hr after labeling. Immunoblotting/fluorography of 2-dimensional gels with monoclonal antibodies to phosphorylated and unphosphorylated NF proteins identified the high-molecular-weight NF subunit in various stages of processing in the DRG between 1 and 48 hr after labeling. The precursor to NF200 migrated on 2-dimensional PAGE as a 160 kDa protein with a pI of about 7.2. During the next 48 hr, the migration of this protein progressively changed to the mature pattern of 200 kDa and a pI of about 5.2. The 145 and 68 kDa NF proteins exhibited very little change in migration on gels during this same interval. Dephosphorylation of mature NF proteins with E. coli alkaline phosphatase regenerated the 160 kDa precursor, confirming that phosphorylation was the main posttranslational mechanism involved in the maturation of newly synthesized high-molecular-weight NF protein. Detergent extraction of labeled DRGs suggested that the 160 kDa NF protein was present in assembled neurofilaments. Immunohistochemical experiments with monoclonal antibodies were performed to explore the intracellular location of phosphorylated and unphosphorylated high-molecular-weight NF protein. Analysis revealed that neuronal cell bodies, as well as short initial segments of DRG axons located within the ganglion, contained unphosphorylated NF protein, while axons in the distal nerve contained mature, phosphorylated NF200. These findings provide support for a model in which posttranslational processing of the 160 kDa precursor occurs in the initial axonal region of DRG cells after the assembled NFs have left the cell body and begun axonal transport.
D000818:Animals; D001369:Axons / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005727:Ganglia, Spinal / Q000032:analysis*; D006651:Histocytochemistry; D007124:Immunoenzyme Techniques; D007381:Intermediate Filament Proteins / Q000032:analysis* / Q000302:isolation & purification; D008297:Male; D008970:Molecular Weight; D016900:Neurofilament Proteins; D010525:Peripheral Nerves / Q000032:analysis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins