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

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.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

“…The fact that the transporters presumably work against a concentration gradient raises the question of the driving force, such as the possibility of a counter exchange with a gradient in the opposite direction. Segregated NAD ϩ pools have also been demonstrated in other organelles such as peroxisomes, the Golgi apparatus, and endoplasmic reticulum (30). So far, only for human peroxisomes, a membrane protein with described NAD ϩ transporter activity could be identified (38).…”

“…Both transporters mediated a strong elevation of mitochondrial NAD ϩ levels, as conveniently monitored by the PARAPLAY-based detection system, which exploits intramitochondrial poly(ADP-ribose) (PAR) formation as readout (23,30). None of the human SLC25 proteins tested had such an effect.…”

“…1A, expression of these transporters resulted in the expected localization of the proteins, as indicated by their co-localization with the mitochondria-specific dye MitoTracker. To determine whether overexpression of these transporters in human cells had an influence on mitochondrial NAD ϩ contents, we made use of the previously established PARAPLAY system (23,30). This system is based on the accumulation of immunodetectable PAR mediated by mitoPARP, a mitochondrially targeted fusion protein consisting of an enhanced GFP (EGFP) portion and the catalytic domain of PARP1, which converts mitochondrial NAD ϩ to PAR in a concentration-dependent manner (23,29).…”

NAD+/NADH and skeletal muscle mitochondrial adaptations to exercise

“…More recently, the compartmentation of NAD ϩ , which was originally suggested by Ragland and Hackett (146), has been extrapolated from the localization of enzymes in the NAD ϩ consuming, biosynthetic, and salvage pathways and the use of innovative molecular biology techniques (11,12,84,97,144,190). Thus, Dölle et al (43) used the novel PAR Assisted Protein Localization AssaY (PARAPLAY) in HeLa S3 cells, in which they targeted the catalytic domain of PARP1 (which consumes NAD ϩ ) to various cellular compartments. The idea behind this method is that if NAD ϩ is present in the compartment to which PARP1 is targeted, then PAR will accumulate and can be detected by immunocytochemistry (43).…”

“…Thus, Dölle et al (43) used the novel PAR Assisted Protein Localization AssaY (PARAPLAY) in HeLa S3 cells, in which they targeted the catalytic domain of PARP1 (which consumes NAD ϩ ) to various cellular compartments. The idea behind this method is that if NAD ϩ is present in the compartment to which PARP1 is targeted, then PAR will accumulate and can be detected by immunocytochemistry (43). Using PARAPLAY, NAD ϩ was found in mitochondria (specifically the matrix but not intermembrane space) and peroxisomes, and surprisingly to the endoplasmic reticulum (ER) and Gogli complex (43,112).…”