Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons.
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)
Schmied
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Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Huang
C C
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Zhang
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Ambron
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Ambron
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D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001048:Aplysia; D001370:Axonal Transport; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005724:Ganglia / Q000378:metabolism / Q000502:physiology; D006657:Histones / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009420:Nervous System / Q000378:metabolism; D009424:Nervous System Physiological Phenomena; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism; D056489:Nucleoplasmins; D009865:Oocytes; D010750:Phosphoproteins; D021382:Protein Sorting Signals / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D014981:Xenopus
D000952:Antigens, Polyomavirus Transforming; D006657:Histones; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012709:Serum Albumin; D006735:Horseradish Peroxidase