Analysis of intracellular translocation of choline acetyltransferase by bioimaging

胆碱乙酰转移酶（Choline Acetyltransferase，ChAT）是乙酰胆碱的合成酶，在多种动物中存在多种ChAT mRNA亚型。由于这些同种型大多是非编码剪接变体，只有约68 kDa的同源ChAT蛋白已被证明在体内产生。最近的证据表明，存在一种蛋白质编码的ChAT mRNA的剪接变体，它缺乏大鼠ChAT基因的外显子6-9。编码的蛋白质被指定为外周型的ChAT（pChAT），因为其在外周神经系统中的优先表达，通过Western印迹和免疫组织化学证实。然而，pChAT的功能意义尚不清楚。为了获得这个问题的线索，我们在活的人胚肾细胞（HEK 293细胞）和PC-12细胞中使用绿色荧光蛋白（GFP）检查了pChAT和公知的普通型ChAT（cChAT）之间的细胞内运输的可能差异。共聚焦激光扫描显微镜显示pChAT-GFP在细胞质中可检测到，但在细胞核中不可检测到，而cChAT-GFP在细胞质和细胞核中均被发现。在用核输出途径抑制剂来普霉素B处理后，pChAT-GFP在细胞质和细胞核中都可检测到，表明pChAT可以易位到细胞核。相反，来普霉素B处理似乎不影响核内cChAT-GFP的含量。与蛋白激酶C抑制剂孵育后，增加的积累pChAT-GFP，但不cChAT-GFP发生在细胞核中。这些结果清楚地表明，pChAT不同于cChAT的细胞内运输，可能反映了pChAT和cChAT之间的生理作用的差异。

Choline acetyltransferase(ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type(pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type(cChAT) using green fluorescent protein(GFP) in living human embryonic kidney cells (HEK293 cells) and PC-12 cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.