Role of mitochondrial complex III/IV in the activation of transcription factor Rst2 in Schizosaccharomyces pombe

Abstract

Mitochondria play essential roles in eukaryotic cells for glucose metabolism to produce ATP. In Schizosaccharomyces pombe, transcription factor Rst2 can be activated upon glucose deprivation. However, the link between Rst2 and mitochondrial function remains elusive. Here, we monitored Rst2 transcriptional activity in living cells using a Renilla luciferase reporter system, and found that inhibition of mitochondrial complex III/IV caused cells to produce reactive oxygen species (ROS) and nitric oxide (NO), which in turn activated Rst2. Furthermore, Rst2‐GFP was observed to translocate from cytoplasm to nucleus upon mitochondrial complex III/IV inhibitors treatment, and deletion of genes associated with complex III/IV resulted in delayed process of Rst2‐GFP nuclear exportation under glucose‐rich condition. In particular, we found that Rst2 was phosphorylated following the treatment of complex III/IV inhibitors or SNAP. Altogether, our findings suggest that mitochondrial complex III/IV participates in the activation of Rst2 through ROS and NO generation in Schizosaccharomyces pombe.

Live‐cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe

“…Thus, this type of reporter allows us to estimate kinase activity based on the ratio of cytoplasmic to nuclear fluorescence intensity (C/N ratio). To construct a KTR‐based PKA biosensor for fission yeast, we focused on the transcription factor Rst2, which is directly phosphorylated by PKA and exhibits nucleocytoplasmic shuttling in a PKA activity‐dependent manner (Figure 1c) (Gupta et al, 2011b; Higuchi et al, 2002; Jiang et al, 2021; Koike et al, 2012). Since overexpression of full‐length Rst2 induces growth defects in fission yeast cells (Koike et al, 2012; Takenaka et al, 2018), we deleted the N‐terminus of Rst2 up to 130 amino acids (a.a.), which contains the two Cys 2 His 2 ‐type zinc finger motifs (Kunitomo et al, 2000).…”

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeastSchizosaccharomyces pombe

“…To construct a KTR-based PKA biosensor for fission yeast, we focused on the transcription factor Rst2, which is directly phosphorylated by PKA and exhibits nucleocytoplasmic shuttling in a PKA activity-dependent manner (Fig. 1C) (Higuchi, Watanabe, and Yamamoto 2002; Gupta et al 2011b; Jiang et al 2021; Koike et al 2012). Since overexpression of full-length Rst2 induces growth defects in fission yeast cells (Takenaka et al 2018; Koike et al 2012), we deleted the N-terminus of Rst2 up to 130 amino acids (a.a.), which contains the two Cys 2 His 2 -type zinc finger motifs (Kunitomo et al 2000).…”

Phosphoinositide-Dependent Protein Kinases Regulate Cell Cycle Progression Through the SAD Kinase Cdr2 in Fission Yeast

“…The fission yeast Schizosaccharomyces pombe ( S. pombe ) has been known for years to be an excellent model system for the investigation of the fundamental regulations and control mechanisms of various cellular processes, due to its high homology with mammals and advantages in gene manipulation ( Forsburg, 2005 ; Liu et al, 2018 , 2020 ; Jiang G. et al, 2021 ; Vyas et al, 2021 ). Ksg1 ( k inase responsible for s porulation and g rowth 1), which is essential for the cell viability in fission yeast, is a homologous protein of mammalian PDK1 ( Niederberger and Schweingruber, 1999 ).…”