THE MECHANISM OF NUCLEAR TRANSPORT OF NATURAL OR ARTIFICIAL TRANSPORTSUBSTRATES IN DIGITONIN-PERMEABILIZED CELLS

Characterization of nuclear protein transport in digitonin-permeabiliz ed cells revealed that the number of the nuclear localization signal s equences (NLS) within the transport substrate basically influences the mechanism of the transport reaction. Phycoerythrine-NLS transport sub strate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methy ltransferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenous ly added cytosolic protein. All the characteristic properties of in vi vo nuclear transport are faithfully reproduced with these transport su bstrates: (i) the transport requires a functional NLS in the transport ed protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear locali zation peptide efficiently competes for transport, while the transport -incompetent mutant peptide does not; and (iv) wheat germ agglutinin i nhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or ineff icient extraction of the cytosolic proteins during the permeabilizatio n of the cells. The nuclear transport was proportional to the number o f conjugated NLSs. Nuclear transport of phycoerythrine carrying 7-8 co njugated NLSs/subunit required the;addition of exogenous cytosolic pro teins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport. Nuclear transport with different c ombinations of transport substrates further supported the assumption t hat distinct transport mechanisms operate for different substrates. Fr om a mixture of PE-NLS(7-8) and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the a bsence of exogenous cytosol, while Bsp methyltransferase-NLS was effic iently transported. Exogenous cytosol promoted the nuclear transport o f the highly conjugated substrate.