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© The Rockefeller University Press, 0021-9525/1997//1185 $5.00
The Journal of Cell Biology, Volume 136, Number 6, , 1997 1185-1199

Article

In Vivo Dynamics of Nuclear Pore Complexes in Yeast



Mirella Bucci and Susan R. Wente

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

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 not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to 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 different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, 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 point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole; F.R., fluorescence ratio; GLFG, glycine-leucine-phenylalanine-glycine; NPC, nuclear pore complex; NUP, nucleoporin; SC, synthetic complete medium.

We are indebted to J. Waddle and J. Cooper for advice in monitoring GFP in live yeast cells, and to J. Waddle and G. Baxter for writing the computer macros for collecting and analyzing the data. We thank M. Rose for the kar1-1 strain; R. Murphy for the gle2-1 strain; L. Riles, J. Waddle, R. Heim, and R. Tsien for plasmids; and J. Cooper, J. Waddle, K. Wilson, M. Winey, and colleagues in the Wente laboratory for insightful discussion and comments on the manuscript.

Address all correspondence to Susan R. Wente, Department of Cell Biology and Physiology, Box 8228, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: (314) 362-2713. Fax: (314) 362-7463. e-mail: swente{at}cellbio.wustl.edu


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