Functional analysis of a novel intracellular calcium-binding protein peflin

Peflin是penta-EF-hand （PEF）钙结合蛋白家族的新成员，该家族包括ALG-2（凋亡相关基因2）、calpain和sorcin。我们制备了一种特异性的单克隆抗体来识别peflin。通过免疫沉淀和Western blot分析，发现peflin和ALG-2形成异源二聚体，以钙依赖的方式解离。peflin定位于细胞质，而ALG-2定位于细胞质和细胞核。Peflin可能通过异源二聚化调节ALG-2的功能。我们在细胞黏菌盘基钢霉（Dictyostelium disideum）中发现了PEF蛋白，盘基钢霉是一种模式生物，在其生命周期中表现出从单细胞形式到多细胞形式的转化。系统发育分析表明，ALG-2和/或peflin在高等和低等真核生物中是保守的，普遍存在，而calpain和sorcin是较新的进化产物。通过酵母双杂交筛选，分离到ALG-2、peflin、Alix和annexin XI等与ALG-2相互作用的蛋白。膜联蛋白n端Gly/Tyr/Pro-rich区域的序列与Alix的c端区域相似，表明这些区域存在共同的结合基序

Peflin is a new member of the penta-EF-hand (PEF) calcium-binding protein family that includes ALG-2 (apoptosis linked gene 2), calpain, and sorcin. We prepared a specific monoclonal antibody that recognized peflin. By immunoprecipitation followed by Western blot analysis, peflin and ALG-2 have been found to form a heterodimer that dissociates in a calcium-dependent manner. While peflin is localized to cytoplasm, ALG-2 is localized in both cytoplasm and nuclei. Peflin may regulate function of ALG-2 by heterodimerization. We identified PEF proteins in the cellular slime mold Dictyostelium discoideum, a model organism that displays transformation from unicellular form to multicellular form during its life cycle. Phylogenetic analysis has revealed that ALG-2 and/or peflin are conserved and present ubiquitously in higher and lower eukaryotes while calpain and sorcin are rather new in evolution. By the yeast two-hybrid screening, ALG-2, peflin, Alix and annexin XI were isolated as ALG-2-interacting proteins. The N-terminal Gly/Tyr/Pro-rich region of annexin has a sequence similar to that of the C-terminal region of Alix, suggesting presence of a common binding motif in these regions