DEPLETION OF CALCIUM FROM THE LUMEN OF ENDOPLASMIC-RETICULUM REVERSIBLY INHIBITS PASSIVE DIFFUSION AND SIGNAL-MEDIATED TRANSPORT INTO THE NUCLEUS

Nuclear pore complexes provide channels for molecular transport across the nuclear envelope. Translocation of most proteins and RNAs through the pore complex is mediated by signal- and ATP-dependent mechanisms, while transport of small molecules is accomplished by passive diffusi on. We report here that depletion of calcium from the lumen of the end oplasmic reticulum and nuclear envelope with ionophores or the calcium pump inhibitor thapsigargin rapidly and potently inhibits signal medi ated transport of proteins into the nucleus. Lumenal calcium depletion also inhibits passive diffusion through the pore complex. Signal-medi ated protein import and passive diffusion are rapidly restored when th e drugs depleting lumenal calcium are removed and cells are incubated at 37 degrees C in calcium-containing medium. These results indicate t hat loss of calcium from the lumen of the endoplasmic reticulum and nu clear envelope reversibly affects properties of pore complex component s located on the nuclear/cytoplasmic membrane surfaces, and they provi de direct functional evidence for conformational flexibility of the po re complex. These methods will be useful for achieving reversible inhi bition of nucleocytoplasmic trafficking for in vivo functional studies , and for studying the structure of the passive diffusion channel(s) o f the pore complex.