The organization of the core proteins of the yeast spindle pole body.
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.
Muller
Eric G D
EG
Department of Biochemistry, University of Washington, Seattle, WA 98195-7350, USA. emuller@u.washington.edu
Snydsman
Brian E
BE
Novik
Isabella
I
Hailey
Dale W
DW
Gestaut
Daniel R
DR
Niemann
Christine A
CA
O'Toole
Eileen T
ET
Giddings
Tom H
TH
Sundin
Bryan A
BA
Davis
Trisha N
TN
D002147:Calmodulin / Q000737:chemistry; D002148:Calmodulin-Binding Proteins; D002502:Centrioles / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D003598:Cytoskeletal Proteins; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005656:Fungal Proteins; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008962:Models, Theoretical; D009687:Nuclear Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D008941:Spindle Apparatus
C505601:CMD1 protein, S cerevisiae; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; C485182:Cyan Fluorescent Protein; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C505600:Spc29 protein, S cerevisiae; D049452:Green Fluorescent Proteins