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MBC in Press, published online ahead of print January 12, 2004
Mol. Biol. Cell 10.1091/mbc.E03-09-0708

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Submitted on September 30, 2003
Revised on December 16, 2003
Accepted on December 18, 2003

The Differential Roles of Budding Yeast Tem1p, Cdc15p, and Bub2p Protein Dynamics in Mitotic Exit

Jeffrey N. Molk1, Scott C. Schuyler2, Jenny Y. Liu3, James G. Evans4, E. D. Salmon1, David Pellman3, and Kerry Bloom1*

1 Department of Biology, University of North Carolina, Chapel Hill, NC 27599
2 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, BioLabs 3000, Cambridge, MA 02138
3 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
4 Whitehead Institute, Massachusetts Institute of Technology, Cambridge MA 02142

* Corresponding author. E-mail address: kerry_bloom{at}unc.edu.

In the budding yeast S. cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the Mitotic Exit Network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in midto late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, Fluorescence Recovery After Photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.




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