Roberts P M, Goldfarb D S
Department of Biology, University of Rochester, New York 14627, USA.
Methods Cell Biol. 1998;53:545-57. doi: 10.1016/s0091-679x(08)60894-8.
We have described a direct fluorescence assay to measure the relative rates of NLS-directed import and passive export of an NLS-GFP fusion protein in yeast. The design and construction of the reporter GFP fusion, its spectral qualities, size, use of inducible promoters, and the choice of NLS, are variables that could extend the method's utility. Future applications will almost certainly demand the quantification of transport rates in single cells using image analysis techniques. As is the case whenever cellular processes are studied in vivo, the in vivo nuclear trafficking properties of NLS-GFP are complicated and poorly understood. Some will be attracted to NLS-GFP kinetic assays simply because so little is known about the function and regulation of the transport apparatus in living cells. At the same time, the uncertainties that accompany in vivo work necessarily prevent the rigorous interpretation of data, which biochemists expect from experiments performed in vitro using highly purified enzymes.
