Department of Animal Science, School of Agriculture, Chinese Culture University, Yang Ming Shan, Taipei, Taiwan.
Mol Cell Biochem. 2012 Jan;360(1-2):121-31. doi: 10.1007/s11010-011-1050-8. Epub 2011 Sep 11.
The Rnf33/Trim60 gene is temporally transcribed in the preimplantation embryo before being silenced at the blastocyst stage but Rnf33 expression is detected in adult testis of the mouse. The putative RNF33 protein is a tripartite motif (TRIM)/RBCC protein composed of a typical RING zinc finger, a B-box 2, two α-helical coiled-coil segments, and a B30.2 domain. As a first step towards the elucidation of the biologic function of RNF33, we aimed in this study to elucidate proteins that associate with RNF33. RNF33-interacting proteins were first derived by the yeast two-hybrid system followed by co-immunoprecipitation assays. Interacting domains were determined by deletion mapping in genetic and biochemical analyzes. RNF33 was shown to interact with the kinesin-2 family members 3A (KIF3A) and 3B (KIF3B) motor proteins in the heterodimeric form known to transport cargos along the microtubule. Domain mapping showed that the RB and B30.2 domains of RNF33 interacted with the respective carboxyl non-motor domains of KIF3A and KIF3B. Since RNF33 interacted with the carboxyl-terminal tail of the KIF3A-KIF3B heterodimer, the motor head section of KIF3A-KIF3B was free and available for association with designated cargo(s) and movement along the microtubule. Data also suggest that RNF33 most likely interacted with KIF3A-KIF3B independent of the adaptor kinesin-associated protein KAP3. This study is a first demonstration of a TRIM protein, namely RNF33, that interacts with the kinesin molecular motors possibly contributing to kinesin-dependent mobilization of specific cargo(s) along the microtubule in the testis of the mouse.
Rnf33/Trim60 基因在胚胎植入前被转录，然后在囊胚阶段被沉默，但在小鼠的成年睾丸中检测到 Rnf33 的表达。假定的 RNF33 蛋白是一种三联基序（TRIM）/RBCC 蛋白，由一个典型的 RING 锌指、B 盒 2、两个 α-螺旋卷曲螺旋片段和 B30.2 结构域组成。作为阐明 RNF33 生物学功能的第一步，我们旨在本研究中阐明与 RNF33 相关的蛋白质。首先通过酵母双杂交系统获得 RNF33 的相互作用蛋白，然后进行共免疫沉淀测定。通过遗传和生化分析中的缺失作图确定相互作用结构域。结果表明，RNF33 以异二聚体形式与驱动蛋白-2 家族成员 3A（KIF3A）和 3B（KIF3B）马达蛋白相互作用，该异二聚体已知可将货物沿微管运输。结构域作图表明，RNF33 的 RB 和 B30.2 结构域与 KIF3A 和 KIF3B 的相应羧基非马达结构域相互作用。由于 RNF33 与 KIF3A-KIF3B 异二聚体的羧基末端尾巴相互作用，因此 KIF3A-KIF3B 的马达头部部分是自由的，可与指定的货物（s）结合并沿微管移动。数据还表明，RNF33 很可能与 KIF3A-KIF3B 相互作用，而不依赖于接头驱动蛋白相关蛋白 KAP3。本研究首次证明了一种 TRIM 蛋白，即 RNF33，它与驱动蛋白分子马达相互作用，可能有助于在小鼠的睾丸中沿微管运动特定货物（s）的驱动蛋白依赖性动员。
