Balasubramanian M K, Hirani B R, Burke J D, Gould K L
Department of Cell Biology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232.
J Cell Biol. 1994 Jun;125(6):1289-301. doi: 10.1083/jcb.125.6.1289.
The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F-actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.
裂殖酵母粟酒裂殖酵母通过中间分裂进行分裂，并且与许多高等真核细胞一样，胞质分裂需要F-肌动蛋白收缩环发挥功能。在粟酒裂殖酵母中，已鉴定出一类胞质分裂缺陷但DNA复制、有丝分裂或隔膜合成无缺陷的cdc-突变体。在本文中，我们展示了其中一个突变体cdc3-124的特征。温度转换实验表明，cdc3突变体无法形成F-肌动蛋白收缩环。我们对cdc3进行了分子克隆，并利用cdc3+基因组DNA通过体内同源重组创建了一个携带cdc3缺失突变的菌株。携带cdc3缺失等位基因的细胞无法存活。它们在胞质分裂时停滞细胞周期，不形成收缩环。对cdc3+基因的DNA序列分析表明，它编码肌动蛋白单体结合蛋白——肌动蛋白结合蛋白。鉴于最近对肌动蛋白结合蛋白的研究，我们提出Cdc3-肌动蛋白结合蛋白通过催化F-肌动蛋白收缩环的形成在胞质分裂中发挥重要作用。与这一观点一致的是，我们观察到Cdc3-肌动蛋白结合蛋白定位于细胞中F-肌动蛋白收缩环形成的中间区域，并且它对于F-肌动蛋白环的形成至关重要。过量产生Cdc3-肌动蛋白结合蛋白的细胞会变长，呈哑铃状，并在胞质分裂时停滞，没有任何可检测到的F-肌动蛋白染色。通过引入携带肌动蛋白编码基因act1+的多拷贝质粒，可以缓解Cdc3-肌动蛋白结合蛋白过量产生的这种影响。我们将这些影响归因于肌动蛋白结合蛋白过量时可能对肌动蛋白单体的隔离作用。
