Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, 5008 Bergen, Norway.
Cell Mol Life Sci. 2010 Feb;67(3):433-43. doi: 10.1007/s00018-009-0190-4. Epub 2009 Nov 10.
Poly-ADP-ribose polymerases (PARPs) use NAD(+) as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD(+) detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD(+) pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD(+) and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.
聚 ADP-核糖聚合酶 (PARPs) 使用 NAD(+) 作为底物生成 ADP-核糖的聚合物。我们将人 PARP1 的催化结构域作为分子 NAD(+) 探测器靶向到细胞细胞器中。聚合物的免疫化学检测表明，在线粒体、过氧化物酶体以及出人意料的内质网和高尔基体中存在不同的细胞内亚 NAD(+) 池。聚合物不会在线粒体膜间空间或细胞质中积累。我们证明了这种特定于隔室的 NAD(+) 和聚 ADP-核糖周转适用于建立细胞器内蛋白质定位。对于过表达的蛋白质，具有 PARP 活性的遗传赋予，聚合物的检测表明与细胞质分离，因此细胞器内居留。在线粒体中，聚合物的积累揭示了 PARP 融合蛋白的基质定位。与目前用于亚细胞蛋白质定位的融合标签相比，这在分辨率上有了很大的提高。因此，我们建立了一种新的分子工具，可用于研究细胞内亚 NAD 代谢和蛋白质定位。
