A molecular switch for Cdc48 activity and localization during oxidative stress and aging

Abstract

Control over a healthy proteome begins with the birth of the polypeptide chain and ends with coordinated protein degradation. One of the major players in eukaryotic protein degradation is the essential and highly conserved ATPase, Cdc48 (p97/VCP in mammals). Cdc48 mediates clearance of misfolded proteins from the nucleus, cytosol, ER, mitochondria, and more. Here we dissect the crosstalk between cellular oxidation and Cdc48 activity by identification of a redox-sensitive site, Cys115. By integrating proteomics, biochemistry, microscopy, and bioinformatics, we show that removal of Cys115's redox-sensitive thiol group leads to accumulation of Cdc48 in the nucleus and consequently, results in severe defects in the oxidative stress response, mitochondrial fragmentation, and a decrease in ERAD and sterol biogenesis. We have thus identified a unique redox switch in Cdc48, which may provide a clearer picture of the importance of Cdc48's localization in maintaining a "healthy" proteome during oxidative stress and chronological aging in yeast.

In Vivo Imaging with Genetically Encoded Redox Biosensors

“…Pichia pastoris E GSH roGFP1 [37,197] roGFP1-iE [37,197,198] roGFP1-iL [198] general redox state Redoxfluor [44] Saccharomyces cerevisiae E GSH rxYFP [31,43,46,[199][200][201] roGFP1 [25][26][27][28][29]202,203] roGFP2 [12,19,33,[204][205][206] roGFP [207] eroGFP [35,36,[38][39][40][41][42]208] Grx1-roGFP2 [15,24,32,47,48,[209][210][211][212][213][214] general redox state rxRFP [30] Redoxfluor [44,215] H 2 O 2 HyPer…”

“…Previous research revealed that Cys115 in the Cdc48 N-terminal domain undergoes oxidation during chronological aging which precedes massive thiol modifications emerging after the metabolic diauxic shift [454]. A paper by Radzinkski et al provides experimental evidence that Cys115 serves as a redox sensor crucial for sustaining Cdc48 cytosolic localization under conditions of oxidative stress, including that observed during senescence [48]. In this work, Grx1-roGFP2 allowed cellular populations with different E GSH to be isolated for investigation of the effect of the Cys115Ser substitution on their growth.…”

“…Moreover, the mass spectrometry analysis of Cdc48 interactom suggested that a set of antioxidant proteins are either misfolded substrates of Cdc48 or that they regulate the redox homeostasis of Cdc48-associated proteins. The authors drew attention to the fact that since mutations in VCP are connected to several human diseases [455], redox metabolism of affected cells should be carefully investigated in future research [48].…”

“…In a cell population, the GEFIs signals reflect the state of each individual member of the group rather than the averaged data. This property paves the way for implementation of sorting algorithms [47][48][49] and allows visualization of heterogeneity in the neighboring regions [49][50][51][52]. 4.…”