Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks

Abstract

Repair of DNA interstrand crosslinks involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that interstrand crosslink-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA, and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mitochondrial DNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we interrogated the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains 1 mitochondrial targeting sequence and 2 nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the mitochondrial targeting sequence. Further analysis revealed that Pso2 is a dual-localized interstrand crosslink repair protein; it can be imported into both nucleus and mitochondria and that genotoxic agents enhance its abundance in the latter. While mitochondrial targeting sequence is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the 2 nuclear localization signal motifs are functionally redundant. Ablation of mitochondrial targeting sequence abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both nuclear localization signal motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between mitochondrial targeting sequence and nuclear localization signal motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 into 2 DNA-bearing organelles is discussed.

SUMOylation of yeast Pso2 enhances its translocation and accumulation in the mitochondria and suppresses methyl methanesulfonate‐induced mitochondrial DNA damage

“…To test this idea, Pso2 ∆ cells expressing MTS::mCherry with either the WT Pso2::GFP or Pso2 K97R,K575R ::GFP variant were grown in the absence or presence of 0.3% MMS. Consistent with previous work (Somashekara & Muniyappa, 2022), mCherry fused to the MTS localized exclusively to the mitochondria. As expected, we observed that the WT Pso2::GFP and Pso2 K97R,K575R ::GFP variant colocalized with DAPI‐stained nuclei in both untreated and MMS‐treated cells.…”

“…As a first step to addressing these open questions, we constructed a plasmid (p416‐ Pso2 ::FLAG), in which the Pso2 gene was fused to one copy of the FLAG coding sequence to the C‐terminus. Our prior work demonstrated that ectopic expression of Pso2::1xFLAG protected Pso2Δ cells from cisplatin‐induced cell death and that its subcellular localization was similar to the wild type (WT) (Somashekara & Muniyappa, 2022).…”

“…Western blot (WB) analysis of eluted proteins from the anti‐FLAG antibody matrix revealed the presence of three distinct immunoreactive bands, including an intense, slow‐migrating 105‐kDa species and two faint, faster‐migrating bands with molecular weights (MWs) of 78 and 68‐kDa, respectively (Figure 1a). While the 78 and 68‐kDa protein bands represent the native and processed forms of Pso2, respectively (Somashekara & Muniyappa, 2022), the identity of the 105 kDa species remained unknown. We hypothesized that the 105‐kDa species might be a post‐translationally modified species of native Pso2.…”

“…Next, we asked whether SUMOylation per se modulates the subcellular localization of Pso2. To address this, we first compared the localization and relative abundance of ectopically expressed WT Pso2::GFP and SUMOylation‐defective Pso2::GFP (Pso2 K97R,K575R ::GFP) variant in the nuclei of MMS‐treated and untreated cells as previously described (Somashekara & Muniyappa, 2022). In untreated cells, we observed that WT Pso2::GFP colocalized with the DAPI‐stained nucleus, with diffuse Pso2::GFP fluorescence throughout the cytoplasm (Figure 6a).…”

“…The plasmid expressing GFP-tagged Pso2 was created in two steps as previously described (Somashekara & Muniyappa, 2022).…”