Hofmann C, Cheeseman I M, Goode B L, McDonald K L, Barnes G, Drubin D G
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.
J Cell Biol. 1998 Nov 16;143(4):1029-40. doi: 10.1083/jcb.143.4.1029.
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.
在本文中，我们描述了两种新型的、对有丝分裂纺锤体至关重要的蛋白质Duo1p和Dam1p的鉴定及特性。Duo1p是因其过表达导致有丝分裂缺陷和有丝分裂停滞而被分离出来的。通过免疫荧光、免疫电子显微镜以及用绿色荧光蛋白（GFP）标记，Duo1p定位于核内纺锤体微管和纺锤极体。温度敏感型duo1突变体因短纺锤体而停滞。这种停滞依赖于有丝分裂检查点。Dam1p通过双杂交分析被鉴定为一种与Duo1p结合的蛋白质。通过在酵母中表达GFP-Dam1p融合蛋白，Dam1p在体内也被证明与核内纺锤体微管和纺锤极体相关。与Duo1p一样，Dam1p的过量产生也导致有丝分裂缺陷。生化实验表明Dam1p以微摩尔亲和力直接与微管结合。我们认为Dam1p可能将Duo1p定位于核内微管和纺锤极体，以提供有丝分裂所需的一种此前未被认识的功能。
