Asakura T, Sasaki T, Nagano F, Satoh A, Obaishi H, Nishioka H, Imamura H, Hotta K, Tanaka K, Nakanishi H, Takai Y
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.
Oncogene. 1998 Jan 8;16(1):121-30. doi: 10.1038/sj.onc.1201487.
We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method. The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.
我们利用¹²⁵I标记的F-肌动蛋白印迹覆盖法，通过连续柱层析从酿酒酵母的可溶部分中纯化出一种新型肌动蛋白丝（F-肌动蛋白）结合蛋白。该纯化蛋白在SDS-聚丙烯酰胺凝胶电泳上显示最小分子量约为140 kDa，我们将其命名为ABP140。用ABP140的部分氨基酸序列在酿酒酵母基因组数据库中进行搜索，结果显示ABP140基因（ABP140）的开放阅读框对应于通过+1移码与YOR240W融合的YOR239W。编码的蛋白质由628个氨基酸组成，计算分子量为71484。重组蛋白与F-肌动蛋白相互作用，并显示出将F-肌动蛋白交联成束的活性。间接免疫荧光研究表明，在完整细胞中ABP140与皮质肌动蛋白斑块和细胞质肌动蛋白电缆共定位。然而，通过基因破坏消除ABP140并未对细胞生长产生有害影响，也未影响F-肌动蛋白的组织。这些结果表明，ABP140对于细胞生长不是必需的，但可能参与出芽酵母中F-肌动蛋白的重组。
