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Regulation of functional nuclear pore size in fibroblasts

¹ Department of Anatomy and Cell Biology, and
² Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA

*Author for correspondence (e-mail: feldherr{at}anatomy.med.ufl.edu)

Accepted September 12, 2001

Protein-NLS-coated gold particles up to approximately 250 Å in diameter are transported through the nuclear pores in normal, proliferating BALB/c 3T3 cells. This size can increase or decrease, depending on cellular activity. It has been suggested that increases in functional pore size are related to a reduction in the amount of available p53. To further test this hypothesis, we investigated the effects of cycloheximide and pifithrin-, which inhibits p53-dependent transcriptional activation, on nuclear transport. After 3 hours in cycloheximide, there was a significant increase in the size of the gold particles that entered the nucleoplasm. When the incubation period was extended to 6 hours or longer, transport capacity returned to the control level. By using proteasome inhibitors, it was shown that the cycloheximide-dependent increase in functional pore size was due to the inhibition of protein synthesis, consistent with the fact that p53 is a short-lived protein, and requires the activity of at least two different factors. Although cycloheximide increases the functional diameter of the channel available for signal-mediated transport by approximately 60 Å, it had no significant effect on either the import rate of small NLS-containing substrates (FITC-BSA-NLS), or passive diffusion of fluorescent-labeled proteins across the envelope. This suggests that changes in transport capacity were not caused by an increase in overall pore diameter but instead are due to a transient increase in pore size that accompanies signal-mediated transport. Pifithrin- also caused an increase in functional pore diameter without altering the import rate of FITC-BSA-NLS, providing further support for the view that p53 can initiate changes in nuclear transport capacity.

Key words: Nuclear pore complex, Nuclear transport, p53