Journal of Neuroscience, Vol 7, 2510-2521, Copyright © 1987 by Society for Neuroscience
Characterization of posttranslational processing of the mammalian high- molecular-weight neurofilament protein in vivo
MM Oblinger
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.
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