Biochem. J. (2013) 454, 217–225 (Printed in Great Britain) doi:10.1042/BJ20121806 217 Hsp104 as a key modulator of prion-mediated oxidative stress in Saccharomyces cerevisiae Kuljit SINGH*, Aliabbas A. SALEH*, Ankan K. BHADRA* and Ipsita ROY* 1 *Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India Maintenance of cellular redox homoeostasis forms an important part of the cellular defence mechanism and continued cell viab- ility. Despite extensive studies, the role of the chaperone Hsp104 (heat-shock protein of 102 kDa) in propagation of misfolded protein aggregates in the cell and generation of oxidative stress remains poorly understood. Expression of RNQ1-RFP in Sacchar- omyces cerevisiae cells led to the generation of the prion form of the protein and increased oxidative stress. In the present study, we show that disruption of Hsp104 in an isogenic yeast strain led to solubilization of RNQ1-RFP. This reduced the oxidative stress generated in the cell. The higher level of oxidative stress in the Hsp104-containing (parental) strain correlated with lower activity of almost all of the intracellular antioxidant enzymes assayed. Surprisingly, this did not correspond with the gene expression ana- lysis data. To compensate for the decrease in protein translation induced by a high level of reactive oxygen species, transcriptional up-regulation takes place. This explains the discrepancy observed between the transcription level and functional enzymatic product. Our results show that in a Hsp104 strain, due to lower oxidative stress, no such mismatch is observed, corresponding with higher cell viability. Thus Hsp104 is indirectly responsible for enhancing the oxidative stress in a prion-rich environment. Key words: antioxidant enzyme, chaperone, Hsp104, oxidative stress, prion, RNQ1. INTRODUCTION In fungal species such as the budding yeast Saccharomyces cerevisiae prions are present as a system of non-Mendelian phenotypic inheritance. Yeast prion is used as a model for studying both the intrinsic properties of the proteins that enable prion propagation and the cellular factors that facilitate prion formation and maintenance [1]. In the non-prion state, these proteins are soluble and are able to perform their molecular function in the cell. Transition to the prion state causes the proteins to aggregate and generally causes a loss-of-function phenotype. Transmission of these aggregates during cell division results in the inheritance of the prion state by daughter cells [1–3]. Due to the self-propagating nature of the prion structure, the associated phenotypes are always dominant. Yeast prion proteins contain N-terminal or C- terminal regions, termed prion domains, that are required for prion formation and propagation [4]. Mechanisms of amyloid formation in yeast and other fungi are similar to mammalian systems. Therefore yeast prions provide easy and efficient experimental assays for studying the factors and conditions influencing amyloid formation and propagation [1,5]. In the yeast S. cerevisiae, several proteins can exist in prion form. The three most well-studied are: (i) a protein of unknown function, RNQ1; (ii) translational termination factor Sup35 (also called eRF3); and (iii) a regulatory protein in the nitrogen metabolism pathway, Ure2. The prion forms of these proteins are termed [PIN + ] (or [RNQ + ]), [PSI + ] and [URE3] respectively [1,5,6]. The yeast prion [RNQ + ]/[PIN + ] is formed by the yeast protein RNQ1. The [RNQ + ] state facilitates the conversion of other proteins into prions in yeast. RNQ1 possesses a C-terminal glutamine/asparagine-rich prion domain which helps it to assemble into amyloid-like fibrils and induces prion formation when fused with the glutamine/asparagine- rich N-terminal domain of Sup35 [7,8]. The N-terminal non- prion domain of RNQ1 regulates [RNQ + ] prion propagation. Interaction between prion aggregates of RNQ1 and soluble Sup35 facilitates [PSI + ] initiation. Initiation of [PSI + ] is enhanced by the temporary overexpression of Sup35p in [RNQ + ] cells [9,10]. Further propagation of [PSI + ] does not require [RNQ + ] prion. Yeast prions depend upon molecular chaperones for efficient maintenance and propagation of prion structures. Many Hsps (heat-shock proteins) are present in the yeast cell and have different roles to play in the formation of amyloid-like fibrils. Expression of certain chaperones prevents the yeast cell from having heritable prions. Some chaperones play a role in the conversion of native proteins into the prion conformation [11,12]. Hsp104 encodes an anti-stress chaperone of the Hsp100 gene family in S. cerevisiae. Hsp104 works in conjunction with other co-chaperones, namely Ssa1 and Ydj1, which have roles to play in the disassembly of protein aggregates that could have accumulated due to stress [13]. Hsp104 chaperone is expressed at very low levels under normal conditions and its expression is induced under stress conditions. Transcriptional activators mediate transcriptional up-regulation. The activators bind to three stress-response elements and two heat-shock elements present in the Hsp104 promoter. Hsp104 mutant strains are unable to propagate the yeast prions [PSI + ], [PIN + ]/[RNQ + ] and [URE3] [1,14,15]. Overexpression and inactivation of Hsp104 help in disaggregation of amyloid fibrils in cells containing [PSI + ] prions [14]. Overexpressed Hsp104 leads to [PSI + ] loss by blocking the incorporation of newly synthesized Sup35p into [PSI + ] complexes or by directly disaggregating the complexes of prions into monomers [12]. The role of Hsp104 in the (dis)assembly of [RNQ + ] is less well understood. Abbreviations used: DCF, dichlorofluorescein; DCFH-DA, dichlorodihydrofluorescein diacetate; DNPH, 2,4-dinitrophenylhydrazine; DTNB, 5,5 ′ -dithiobis- (2-nitrobenzoic acid); Hsp, heat-shock protein; IPOD, insoluble protein deposit; JUNQ, juxtanuclear quality control compartment; MMLV, Moloney murine leukaemia virus; NBT, Nitro Blue Tetrazolium; ROS, reactive oxygen species; SC-LEU, synthetic complete medium without leucine; SOD, superoxide dismutase. 1 To whom correspondence should be addressed (email ipsita@niper.ac.in). c  The Authors Journal compilation c  2013 Biochemical Society Biochemical Journal www.biochemj.org