Journal of Neuroscience, Vol 13, 4064-4071, Copyright © 1993 by Society for Neuroscience
Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons
R Schmied, CC Huang, XP Zhang, DA Ambron and RT Ambron
Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
When the nuclear localization signal peptide (sp) of the SV 40 large T
antigen was coupled to human serum albumin (HSA), rhodaminated (r), and
microinjected into axons of Aplysia neurons in vitro, the rHSA-sp was
conveyed through the axon to the cell body and then into the nucleus
(Ambron et al., 1992). But since rHSA-sp is an artificial construct, we
needed to determine whether naturally occurring nuclear proteins use this
pathway. We therefore injected calf thymus histone H-1 and Xenopus oocyte
nucleoplasmin into axons. By 3 hr both were retrogradely transported and
targeted into the nucleus, though histone H-1 less efficiently than rHSA-sp
or nucleoplasmin. In contrast, neither rHSA, nor rHSA conjugated to a
peptide with a random distribution of basic amino acids, was transported or
imported. To see if proteins that use the pathway remain intact, we coupled
sp to HRP. When injected into varicosities, the HRP-sp was
transported/imported to the nucleus, where it was enzymatically active. A
key issue was to determine whether endogenous proteins use this pathway.
Consequently, axoplasm was extruded from Aplysia nerves and the proteins
were fractionated by size. SDS-PAGE and Western blots showed that two
fractions contained proteins that were recognized by an affinity-purified
antibody to sp: fraction 3 included sp83, and fraction 4 contained sp75. In
addition, these two proteins were found in nuclei isolated from neurons. To
assess transport, the total proteins in the fractions were rhodaminated and
injected into varicosities. Fraction 3, but not fraction 4, contained
protein that was transported through the axon to the nucleus.(ABSTRACT
TRUNCATED AT 250 WORDS)
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