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Originally published as MBC in Press, 10.1091/mbc.E03-09-0708 on January 12, 2004

Vol. 15, Issue 4, 1519-1532, April 2004

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

Jeffrey N. Molk *, Scott C. Schuyler {dagger} {ddagger}, Jenny Y. Liu {dagger}, James G. Evans §, E. D. Salmon *, David Pellman {dagger}, and Kerry Bloom * ¶

* Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599; {dagger} Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115; and § Whitehead Institute, Massachusetts Institute of Technology, Cambridge Massachusetts 02142

Submitted September 30, 2003; Revised December 16, 2003; Accepted December 18, 2003
Monitoring Editor: Anthony Bretscher

In the budding yeast Saccharomyces 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 mid-to-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.

Abbreviations used: CFP, cyan fluorescence protein; DIC, differential interference contrast; dSPB, daughter bound spindle pole body; FRAP, fluorescence recovery after photobleaching; GAP, guanine-activating protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescence protein; MEN, mitotic exit network; mSPB mother bound spindle pole body; SPBs, spindle pole bodies; YFP, yellow fluorescence protein.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03–09–0708. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03–09–0708.

{boxd}<{boxv}< Online version of this article contains supplementary figures and videos. Online version is available at www.molbiolcell.org.

{ddagger} Present address: Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, BioLabs 3000, Cambridge, MA 02138.

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




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