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Vol. 16, Issue 7, 3341-3352, July 2005

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The Organization of the Core Proteins of the Yeast Spindle Pole Body

Eric G.D. Muller ^*, Brian E. Snydsman ^*, Isabella Novik , Dale W. Hailey ^* , Daniel R. Gestaut ^*, Christine A. Niemann ^*, Eileen T. O'Toole , Tom H. Giddings, Jr , Bryan A. Sundin ^*, and Trisha N. Davis ^*

^* Department of Biochemistry, University of Washington, Seattle, WA 98195-7350; Department of Mathematics, University of Washington, Seattle, WA 98195-4350; and Boulder Laboratory for Three-dimensional Fine Structure, University of Colorado, Boulder, CO 80309-0347

Submitted March 16, 2005; Revised April 22, 2005; Accepted April 25, 2005
Monitoring Editor: Sandra Schmid

The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to each other was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis.

Abbreviations used: SPB, spindle pole body; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: NIH/NICHD/CBMB, Building 18, Room 101, 9000 Rockville Pike, Bethesda, MD 20892-5430.

Address correspondence to: Eric G.D. Muller (emuller{at}u.washington.edu).

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