title: Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope.
abstract: The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1's function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31-Cdc31 interactions between Sfi1-Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Seybold
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Menattallah
authors.initials: M
authors.lastname: Elserafy
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Rüthnick
authors.affiliation: Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany Heidelberg Graduate School of Mathematical and Computational Methods for the Sciences, Heidelberg University, 69120 Heidelberg, Germany.
authors.forename: Musa
authors.initials: M
authors.lastname: Ozboyaci
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Annett
authors.initials: A
authors.lastname: Neuner
authors.affiliation: Institute for Anatomy and Cell Biology, Functional Neuroanatomy, Heidelberg University, 69120 Heidelberg, Germany Institute for Physical and Theoretical Chemistry, Johann-Wolfgang-Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Flottmann
authors.affiliation: Institute for Anatomy and Cell Biology, Functional Neuroanatomy, Heidelberg University, 69120 Heidelberg, Germany Institute for Physical and Theoretical Chemistry, Johann-Wolfgang-Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, 69120 Heidelberg, Germany.
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Wade
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany e.schiebel@zmbh.uni-heidelberg.de.
authors.forename: Elmar
authors.initials: E
authors.lastname: Schiebel
mesh_terms: D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D064048:Spindle Pole Bodies / Q000378:metabolism*
chemical_list: C083769:CDC31 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C074559:KAR1 protein, S cerevisiae; D009687:Nuclear Proteins; D012097:Repressor Proteins; C121820:SFI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins