Department of Biochemistry, University of Wisconsin-Madison, WI 53706.
Stowers Institute for Medical Research, Kansas City, MO 64110.
Mol Biol Cell. 2019 Jun 1;30(12):1505-1522. doi: 10.1091/mbc.E19-03-0167. Epub 2019 Apr 10.
Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Å hexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.
中心体和纺锤体极体（SPB）是无膜细胞器，其复制和组装对于形成两极有丝分裂纺锤体是必要的。这些细胞器中主要蛋白质的结构组织和功能作用可以为细胞分裂控制提供重要的见解。Spc42 是一种磷酸化调节蛋白，具有 N 端二聚化卷曲螺旋（DCC），在出芽酵母 SPB 核心中组装成六聚体阵列，在那里它作为 SPB 组装的支架。在这里，我们提供了体外和体内数据来阐明 Spc42 元件的结构排列和生物学作用。晶体结构揭示了 Spc42 中另外两个卷曲螺旋的细节：中央三聚体卷曲螺旋和 C 端反平行 DCC。三个 Spc42 卷曲螺旋和相邻未确定区域对体外脂质单层测定中形成约 145Å 的六聚体晶格以及体内 SPB 复制和组装的贡献揭示了 Spc42 组装的结构和功能冗余。我们提出了一个更新的模型，将这些 Spc42 元件的固有对称性纳入晶格中，从而建立了观察到的六重对称性。该模型对中央 SPB 核心层的组织的影响进行了讨论。
