Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase
Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase
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DOI：
10.1242/jcs.00246
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发表时间：
2003-02-15
影响因子：
4
通讯作者：
Hanover, JA
中科院分区：
文献类型：
--
作者：
Love, DC;Kochran, J;Hanover, JA
O-连接的GlcNAc转移酶（OGT）介导一种新的聚糖依赖性信号传导途径，但对OGT的细胞内靶向知之甚少。我们研究了本地化的OGT免疫荧光显微镜，亚细胞分级和免疫印迹使用高度特异性的亲和纯化的抗血清。除了预期的核定位，我们发现，OGT是高度集中在线粒体。由于线粒体OGT（103 kDa）小于其他隔室中发现的OGT（116 kDa），我们推断它是OGT的两种预测剪接变体之一。这些亚型的N-末端是独特的;较短的形式含有潜在的线粒体靶向序列。我们发现，当表位标记，较短的形式（mOGT，103 kDa）集中在HeLa细胞线粒体，而较长的形式（ncOGT，116 kDa）定位于细胞核和细胞质。mOGT的N-末端对于适当的靶向是必不可少的。虽然mOGT似乎是一种活性转移酶，但O-连接的GlcNAc修饰的底物不会在线粒体中积累。利用免疫电镜和线粒体分离技术，我们发现mOGT与线粒体内膜紧密结合。OGT的线粒体和核质异构体的差异定位表明它们执行独特的细胞内功能。
O-linked GlcNAc transferase (OGT) mediates a novel glycan-dependent signaling pathway, but the intracellular targeting of OGT is poorly understood. We examined the localization of OGT by immunofluorescence microscopy, subcellular fractionation and immunoblotting using highly specific affinity-purified antisera. In addition to the expected nuclear localization, we found that OGT was highly concentrated in mitochondria. Since the mitochondrial OGT (103 kDa) was smaller than OGT found in other compartments (116 kDa) we reasoned that it was one of two predicted splice variants of OGT. The N-termini of these isoforms are unique; the shorter form contains a potential mitochondrial targeting sequence. We found that when epitope-tagged, the shorter form (mOGT; 103 kDa) concentrated in HeLa cell mitochondria, whereas the longer form (ncOGT, 116 kDa) localized to the nucleus and cytoplasm. The N-terminus of mOGT was essential for proper targeting. Although mOGT appears to be an active transferase, O-linked GlcNAc-modified substrates do not accumulate in mitochondria. Using immunoelectron microscopy and mitochondrial fractionation, we found that mOGT was tightly associated with the mitochondrial inner membrane. The differential localization of mitochondrial and nucleocytoplasmic isoforms of OGT suggests that they perform unique intracellular functions.