IN-VIVO DYNAMICS OF NUCLEAR-PORE COMPLEXES IN YEAST

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is no t well defined. To investigate the dynamics of NPCs, we developed a li ve-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin N up49p was fused to the green fluorescent protein (GFP) of Aequorea ea victoria and expressed in nup49 null haploid yeast cells. When the GFP -Nup49p donor cell was mated with a recipient cell harboring only unla beled Nup49p, the nuclei fused as a consequence of the normal mating p rocess, By monitoring the distribution of the GFP-Nup49p, we could ass ess whether NPCs were able to move from the donor section of the nucle ar envelope to that of the recipient nucleus, We observed that fluores cent NPCs moved and encircled the entire nucleus within 25 min after f usion, When assays were done in mutant karl-l strains, where nuclear f usion does not occur, GFP-Nup49p appearance in the recipient nucleus o ccurred at a very slow rate, presumably due to new NPC biogenesis or t o exchange of GFP-Nup49p into existing recipient NPCs, Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures, This has been explained with two diff erent hypotheses: by movement of NPCs through the double nuclear membr anes with subsequent clustering at a central location; or, alternative ly, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this po int, Using the GFP-Nup49p system with a mutant in the NPC-associated f actor Gle2p that exhibits formation of NPC clusters only at 37 degrees C, it was possible to distinguish between these two models for NPC dy namics. GFP-Nup49p-labeled NPCs, assembled at 23 degrees C, moved into clusters when the cells were shifted to growth at 37 degrees C. These results indicate that NPCs can move through the double nuclear membra nes and, moreover, can do so to form NPC clusters in mutant strains, S uch clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation, T his system represents a novel approach for identifying regulators of N PC assembly and movement in the future.