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Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation

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Abstract

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.

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Acknowledgments

Christian Dölle, Marc Niere, and Mathias Ziegler are named as inventors in a pending patent application on this issue. The patent application is owned by Bergen Teknologioverføring AS.

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Authors and Affiliations

  1. Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, 5008, Bergen, Norway

    Christian Dölle, Marc Niere, Emilia Lohndal & Mathias Ziegler

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  1. Christian Dölle
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  2. Marc Niere
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  3. Emilia Lohndal
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  4. Mathias Ziegler
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Corresponding author

Correspondence to Mathias Ziegler.

Additional information

C. Dölle and M. Niere contributed equally to this work.

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Dölle, C., Niere, M., Lohndal, E. et al. Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation. Cell. Mol. Life Sci. 67, 433–443 (2010). https://doi.org/10.1007/s00018-009-0190-4

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  • DOI: https://doi.org/10.1007/s00018-009-0190-4

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