Ysa1 is the major ADPr/OAADPr-metabolizing enzyme in yeast. HPLCs (A_{260 nm} versus time) of 1 mm ADPr/OAADPr/3′-NAADPr were incubated with cell lysates or buffer (50 mm Tris, pH 7.5, 10 mm MgCl₂) controls at 37 °C for 30 min as follows: peak 1, ADPr; peak 2, AMP; peak 3, 3′-OAADPr; peak 4, 2′-OAADPr; peak 5, 3′-NAADPr. A, 1 mm ADPr with wild type cell extract (top panel), Δysa1 cell extract (middle panel), or buffer (bottom panel). B, 1 mm OAADPr with wild type cell extract (top panel), Δysa1 cell extract (middle panel), or buffer (bottom panel). C, 1 mm 3′-NAADPr with wild type cell extract (top panel), Δysa1 cell extract (middle panel), or buffer (bottom panel).

Δysa1 cells show increased resistance to copper-induced stress. A, growth comparison of wild type and Δysa1 cells on YMD plates containing 0 mm CuSO₄ (top panel), 0.3 mm CuSO₄ (middle panel), and 1 mm CuSO₄ (bottom panel). Serial dilutions of each culture were spotted onto YMD plates and grown at 30 °C for 2 days before imaging. B, survival test comparing wild type (WT) and Δysa1 cells under CuSO₄ stress. Top panel, cells treated with 9 mm copper for 24 h and rescued on YPD plates for 2 days. Bottom panel, control cells without CuSO₄ treatment. Comparison of the wild type (top left panel) and Δysa1 cells (top right panel) shows the Δysa1 cells have increased resistance to copper-induced stress.

ADPr/OAADPr interact with glycolytic enzymes. A, positive control for affinity elution. Endogenous Sir2 interacts with OAADPr. Anti-Sir2 Western blot shows the elution profile of wild type yeast cell extract applied to Cibacron blue resin and eluted with the indicated concentrations of OAADPr. B, identification of novel ADPr/OAADPr-interacting proteins. SYPRO Ruby-stained SDS-polyacrylamide gel shows the Cibacron blue elution profile for a wild type cell extract eluted with either OAADPr or ADPr. Indicated bands were in-gel digested with trypsin and identified by LC-MS/MS as phosphoglycerate kinase (band 1), alcohol dehydrogenase (band 2), and glyceraldehyde-3-phosphate dehydrogenase (band 3). C, double-reciprocal plot of GAPDH inhibition by ADPr. Initial rates were determined from the rate of NAD⁺ reduction. Assays were performed in the presence of 1 mm glyceraldehyde 3-phosphate (GAP), and NAD⁺ concentrations ranged from 43 to 286 μm. ADPr exhibits competitive inhibition toward NAD⁺. The following ADPr concentrations were used: 60 (•), 100 (♦), 150 (▪), and 300 μm (▴). Data were fitted to competitive inhibition using Kinetasyst as described under “Experimental Procedures.” Shown here is one representative of three replicated experiments. K_i value of ADPr and K_m value of NAD⁺ were 60.5 ± 15.4 and 17.5 ± 3.1 μm (average ± S.E.) respectively, averaged from three replicate experiments.

Proposed functions for Ysa1 and ADPr/OAADPr in metabolic pathways and cellular redox. ADPr/OAADPr generated from NAD⁺ cleavage are directly hydrolyzed by Ysa1 into AMP. Accumulation of AMP activates glycolysis. Deletion of Ysa1 results in increased cellular ADPr/OAADPr and decreased AMP. As a consequence, ADPr/OAADPr inhibits glycolysis (e.g. GAPDH) and promotes NADPH production from the pentose phosphate pathway. Because NADPH is a major source of cellular reducing power for ROS-detoxifying enzymes, the increase in cellular NADPH strengthens the antioxidative stress response capability of Δysa1 cells. Additionally, increased ADPr/OAADPr in Δysa1 inhibits the mitochondrial electron transport chain through complex I, leading to lower in vivo ROS levels.

Δysa1 cells show increased resistance to H₂O₂. A, halo assays show that Δysa1 cells display increased resistance to H₂O₂ stress versus wild type cells. Wild type (WT) and Δysa1 cells from liquid culture were spread onto YPD plates and then spotted with 5 μl of H₂O₂ (1.5 m, top panel; 2.9 m, middle panel; 4.4 m, bottom panel), and allowed to grow for 1 day before the plates were imaged to determine the amount of cell death, as visualized by the diameter of the “halo,” where cell death occurred (dark area) because of H₂O₂ treatment. The Δysa1 cells (right panels) display smaller halos for all H₂O₂ concentrations tested versus wild type cells (left panels). B, survival assays also show Δysa1 cells display increased resistance to H₂O₂ treatment compared with wild type cells. Graph shows percent cell survival versus time for wild type (diamonds) and Δysa1 (circles) cells, after treatment with 3 mm H₂O₂ for the specified time points.

Endogenous ROS levels and mitochondrial membrane potential ΔΨm decrease in Δysa1 cells and Ysa1 localizes to the mitochondria. A, Δysa1 cells display lower basal levels of ROS. Bar graph shows percent of cells positive for ROS/ethidium, wild type (WT, black bar), and Δysa1 (gray bar). Cells were stained with 5 μg/ml dihydroethidium in phosphate-buffered saline buffer for 15 min and analyzed via flow cytometry. Unstained cells were used as controls for background fluorescence. Results are averages (with standard errors) of three biological replicates. B, Δysa1 cells generate lower mitochondrial membrane potential compared with wild type cells. Cells were stained with rhodamine 123 (Rho123) and analyzed by flow cytometry. Rhodamine 123 signal, which reflects the mitochondrial membrane potential, is plotted as cell number versus averaged intensity signal for three wild type strain biological replicates (black) and three Δysa1 strains (gray) biological replicates. In the upper range (indicated by the black bar), 51% of wild type cells are accounted for where as only 33% Δ ysa1 cells are in the same range. C, Ysa1 (26 kDa) is localized to mitochondria. Western blots show that the TAP-Ysa1 construct (54 kDa) localizes to mitochondria. Cells expressing TAP-Ysa1 were lysed and separated into whole cell, cytoplasm, nuclei, and mitochondrial fractions. Ysa1 was detected using anti-TAP antibody, whereas H3 and Isu1 were used as marker proteins for nuclei and mitochondria, respectively. Top panel, TAP-Ysa1 was detected in whole cell extract and possibly in the nuclei. Bottom panel, strong signal for TAP-Ysa1 was detected in whole cell extract and mitochondria but not in the cytoplasmic fraction. Note, we also detect degraded forms (smaller bands of ∼34 and 26 kDa) of TAP-Ysa1 in both whole cell and mitochondrial samples. The vertical lines indicate where extraneous lanes were removed electronically from the blot; however, the image was not manipulated in any other manner.

Δysa1 cells exhibit higher cellular NADPH concentrations. The cellular concentrations of NADPH in wild type (WT, black bar) and Δysa1 (gray bar) cells were determined by HPLC analysis to be 8 μm for wild type and 14 μm for Δysa1. Results are averages (with standard errors) of three biological replicates.