A nuclear localization signal targets proteins to the retrograde transport system, thereby evading uptake into organelles in aplysia axons.
The turnover of soluble proteins in axons and terminals is effected by replacing used proteins with newly synthesized constituents from the cell body. To investigate this complex process, which is especially important during nerve regeneration, we microinjected proteins into varicosities on axons of Aplysia neurons in vitro. When human serum albumin (HSA) coupled to rhodamine (r) was injected, it initially filled the varicosity; within seconds, however, it began to accumulate in packets and by 15 min was punctate. A similar pattern was observed after injecting soluble proteins from extruded axoplasm. In contrast, when we injected rHSA covalently attached to the SV-40 nuclear localization sequence (sp), the distribution was never punctate and the rHSA-sp was retrogradely transported from the varicosity to the cell body and into the nucleus. Electron microscopy of varicosities injected with HSA-gold showed that >90% of the particles were inside vacuoles and multivesicular bodies. These organelles probably function as storage rather than degradatory sites since they did not contain acid phosphatase. In contrast, when HSAsp-colloidal gold was injected, only 25% of the particles were in organelles. Thus, HSA and resident axonal proteins can be removed from axoplasm by uptake into organelles. The presence of a nuclear localization sequence (the sp) may avoid uptake by providing access to the retrograde transport/nuclear import pathway.
Schmied
R
R
Department of Anatomy and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
Ambron
R T
RT
D000135:Acid Phosphatase / Q000032:analysis; D000418:Albumins / Q000493:pharmacokinetics; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000494:pharmacology; D001048:Aplysia; D001369:Axons / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D017979:Gold Colloid / Q000493:pharmacokinetics; D006801:Humans; D008845:Microinjections; D008854:Microscopy, Electron; D015388:Organelles / Q000201:enzymology*; D021382:Protein Sorting Signals / Q000502:physiology*; D012235:Rhodamines
D000418:Albumins; D000952:Antigens, Polyomavirus Transforming; D017979:Gold Colloid; D021382:Protein Sorting Signals; D012235:Rhodamines; D000135:Acid Phosphatase