Nucleoplasmin uptake by facilitated transport and intranuclear binding.
Specific proteins are selectively translocated into the cell nucleus and accumulated therein, but the molecular mechanisms underlying this fundamental eukaryotic transport process remain obscure. We have employed a new experimental system with notable advantages for resolving protein translocation and accumulation mechanisms. Individual nuclei are isolated from oocytes under mineral oil and conjoined under the oil with either an aqueous bead or a similar volume of oocyte cytoplasm to form closed transport pairs. Using these pairs one can (i) present transportant proteins via the bead or cytoplasm to a minimally disturbed nucleus, (ii) monitor the intactness of the nuclear envelope, and (iii) separate pairs at various times after formation and measure the amount of transportant in each compartment. In addition, it is uniquely possible with these pairs to determine whether or not a transportant's concentration gradient constitutes a chemical activity gradient. This is done by puncturing the envelope, thus eliminating its normal sieving restrictions on diffusion, and measuring the effect on the transportant distributions. We demonstrate that a prototypical nuclear-accumulating protein, nucleoplasmin (Np), is translocated through the nuclear pore complex by a mechanism of facilitated transport, rather than active transport. We further show that Np's high accumulation results from subsequent intranuclear binding. Np's facilitated transport and intranuclear binding are both ATP-dependent, and the latter requires cytoplasmic protein(s).
Vancurova
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Department of Biological Sciences, St. John's University, Jamaica, NY 11439.
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Paine
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D009687:Nuclear Proteins / Q000378:metabolism*; D056489:Nucleoplasmins; D009865:Oocytes / Q000378:metabolism; D010750:Phosphoproteins; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D014982:Xenopus laevis
D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D014909:Wheat Germ Agglutinins; D000255:Adenosine Triphosphate; D005033:Ethylmaleimide