Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol
2021; Springer Nature; Volume: 40; Issue: 16 Linguagem: Inglês
10.15252/embj.2021107913
ISSN1460-2075
AutoresAnna M. Schlagowski, Katharina Knöringer, Sandrine Morlot, Ana Sánchez Vicente, Tamara Flohr, Lena Krämer, Felix Boos, Nabeel Khalid, Sheraz Ahmed, Jana Schramm, Lena Maria Murschall, Per Haberkant, Frank Stein, Jan Riemer, Benedikt Westermann, Ralf J. Braun, Konstanze F. Winklhofer, Gilles Charvin, Johannes M. Herrmann,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle30 June 2021Open Access Source DataTransparent process Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol Anna M Schlagowski Anna M Schlagowski Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Katharina Knöringer Katharina Knöringer orcid.org/0000-0002-1928-2779 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Sandrine Morlot Sandrine Morlot Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Ana Sánchez Vicente Ana Sánchez Vicente Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany Search for more papers by this author Tamara Flohr Tamara Flohr orcid.org/0000-0002-8775-7959 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Lena Krämer Lena Krämer Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Felix Boos Felix Boos orcid.org/0000-0002-1571-9806 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Nabeel Khalid Nabeel Khalid German Research Center for Artificial Intelligence DFKI, Kaiserslautern, Germany Search for more papers by this author Sheraz Ahmed Sheraz Ahmed German Research Center for Artificial Intelligence DFKI, Kaiserslautern, Germany Search for more papers by this author Jana Schramm Jana Schramm Cell Biology, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Lena M Murschall Lena M Murschall Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Per Haberkant Per Haberkant Proteomics Core Facility, EMBL Heidelberg, Heidelberg, Germany Search for more papers by this author Frank Stein Frank Stein orcid.org/0000-0001-9695-1692 Proteomics Core Facility, EMBL Heidelberg, Heidelberg, Germany Search for more papers by this author Jan Riemer Jan Riemer orcid.org/0000-0002-7574-8457 Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Benedikt Westermann Benedikt Westermann orcid.org/0000-0002-2991-1604 Cell Biology, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Ralf J Braun Ralf J Braun orcid.org/0000-0003-4234-8163 Cell Biology, University of Bayreuth, Bayreuth, Germany Neurodegeneration, Danube Private University, Krems/Donau, Austria Search for more papers by this author Konstanze F Winklhofer Konstanze F Winklhofer orcid.org/0000-0002-7256-8231 Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany Search for more papers by this author Gilles Charvin Gilles Charvin orcid.org/0000-0002-6852-6952 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Johannes M Herrmann Corresponding Author Johannes M Herrmann [email protected] orcid.org/0000-0003-2081-4506 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Anna M Schlagowski Anna M Schlagowski Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Katharina Knöringer Katharina Knöringer orcid.org/0000-0002-1928-2779 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Sandrine Morlot Sandrine Morlot Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Ana Sánchez Vicente Ana Sánchez Vicente Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany Search for more papers by this author Tamara Flohr Tamara Flohr orcid.org/0000-0002-8775-7959 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Lena Krämer Lena Krämer Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Felix Boos Felix Boos orcid.org/0000-0002-1571-9806 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Nabeel Khalid Nabeel Khalid German Research Center for Artificial Intelligence DFKI, Kaiserslautern, Germany Search for more papers by this author Sheraz Ahmed Sheraz Ahmed German Research Center for Artificial Intelligence DFKI, Kaiserslautern, Germany Search for more papers by this author Jana Schramm Jana Schramm Cell Biology, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Lena M Murschall Lena M Murschall Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Per Haberkant Per Haberkant Proteomics Core Facility, EMBL Heidelberg, Heidelberg, Germany Search for more papers by this author Frank Stein Frank Stein orcid.org/0000-0001-9695-1692 Proteomics Core Facility, EMBL Heidelberg, Heidelberg, Germany Search for more papers by this author Jan Riemer Jan Riemer orcid.org/0000-0002-7574-8457 Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Benedikt Westermann Benedikt Westermann orcid.org/0000-0002-2991-1604 Cell Biology, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Ralf J Braun Ralf J Braun orcid.org/0000-0003-4234-8163 Cell Biology, University of Bayreuth, Bayreuth, Germany Neurodegeneration, Danube Private University, Krems/Donau, Austria Search for more papers by this author Konstanze F Winklhofer Konstanze F Winklhofer orcid.org/0000-0002-7256-8231 Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany Search for more papers by this author Gilles Charvin Gilles Charvin orcid.org/0000-0002-6852-6952 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Johannes M Herrmann Corresponding Author Johannes M Herrmann [email protected] orcid.org/0000-0003-2081-4506 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany Search for more papers by this author Author Information Anna M Schlagowski1, Katharina Knöringer1, Sandrine Morlot2,3,4,5, Ana Sánchez Vicente6, Tamara Flohr1, Lena Krämer1, Felix Boos1, Nabeel Khalid7, Sheraz Ahmed7, Jana Schramm8, Lena M Murschall9, Per Haberkant10, Frank Stein10, Jan Riemer9, Benedikt Westermann8, Ralf J Braun8,11, Konstanze F Winklhofer6, Gilles Charvin2,3,4,5 and Johannes M Herrmann *,1 1Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany 2Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France 3Centre National de la Recherche Scientifique, UMR7104, Illkirch, France 4Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France 5Université de Strasbourg, Illkirch, France 6Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany 7German Research Center for Artificial Intelligence DFKI, Kaiserslautern, Germany 8Cell Biology, University of Bayreuth, Bayreuth, Germany 9Biochemistry, University of Cologne, Cologne, Germany 10Proteomics Core Facility, EMBL Heidelberg, Heidelberg, Germany 11Neurodegeneration, Danube Private University, Krems/Donau, Austria *Corresponding author. Tel: +49 631 2052406; Fax: +49 631 2052492; E-mail: [email protected] The EMBO Journal (2021)40:e107913https://doi.org/10.15252/embj.2021107913 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation-prone polyQ protein derived from human huntingtin. Expression of Q97-GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97-GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post-translational import of mitochondrial precursor proteins into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate-limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis. SYNOPSIS The accumulation of mitochondrial precursor proteins in the cytosol induces the formation of protein aggregates. Overexpression of mitochondrial import factor Mia40 improves mitochondrial protein import and increases resistance against proteotoxic insults in Saccharomyces cerevisiae. S. cerevisiae mitochondrial import mutants are hypersensitive to cytosolic polyQ proteins. Overexpression of Mia40 prevents the formation of polyQ aggregates in yeast and human cells. Mia40 levels affect the solubility of the cytosolic prion protein Rnq1. Increasing mitochondrial import augments robustness of cytosolic proteostasis. Introduction The accumulation of misfolded or aggregated proteins is frequently observed in stressed cells. Increased levels of misfolded proteins can be the cause, but also the consequence of disease states (for overview, see Chiti & Dobson, 2017; Sontag, Samant et al, 2017; Vaquer-Alicea & Diamond, 2019). Owing to their capability to sequester chaperones and to occupy proteases, misfolded proteins can route metastable or slowly folding polypeptides into aggregates (Gidalevitz, Ben-Zvi et al, 2006; Kim, Hosp et al, 2016). This might induce a calamitous amplifying reaction which finally leads to proteotoxicity and cell death. Cells employ a number of different strategies to avoid the hazardous accumulation of misfolded proteins, including their stabilization and disaggregation by chaperones, their degradation by proteases, and their controlled sequestration into aggregates. Mitochondrial functionality modulates cytosolic protein homeostasis in several ways: (i) Protein aggregates often associate with the mitochondrial outer membrane (Aguilaniu, Gustafsson et al, 2003; Liu, Lillo et al, 2004; Ferri, Cozzolino et al, 2006; Gruber, Hornburg et al, 2018; Suhm, Kaimal et al, 2018; Yablonska, Ganesan et al, 2019); in yeast, this property is used to retain misfolded proteins in the mother in order to prevent their inheritance to daughter cells (Zhou, Slaughter et al, 2011; Mogk & Bukau, 2014; Böckler, Chelius et al, 2017). (ii) Cytosolic aggregates impair the import of precursor proteins from the cytosol into mitochondria (Li, Vande Velde et al, 2010; Napoli, Wong et al, 2013; Yano, Baranov et al, 2014; Cenini, Rub et al, 2016). (iii) Vice versa, mitochondrial precursor proteins in the cytosol challenge proteostasis and sequester chaperones and the ubiquitin–proteasome system (Wrobel, Topf et al, 2015; Boos, Krämer et al, 2019; Poveda-Huertes, Matic et al, 2020). In this context, the hydrophobic carrier proteins of the inner membrane appear to be of specific relevance owing to their hydrophobic nature and specific import mechanism (Williams, Jan et al, 2014; Wang & Chen, 2015). (iv) Modulating mitochondrial activities can induce stress resistance pathways and thereby mitigate the accumulation and toxicity of cytosolic aggregates (Mason, Casu et al, 2013; Labbadia, Brielmann et al, 2017; Sorrentino, Romani et al, 2017; Fessler, Eckl et al, 2020; Guo, Aviles et al, 2020; Straub, Weraarpachai et al, 2021). (v) It was proposed that aggregated cytosolic proteins are disentangled on the mitochondrial surface and imported into the mitochondria to enable their degradation by mitochondrial proteases (Ruan, Zhou et al, 2017; Li, Xue et al, 2019) or removal by mitophagy (Guo, Ma et al, 2012; Hwang, Disatnik et al, 2015; Khalil et al, 2015). The implications of mitochondrial biology for cytosolic proteostasis are even more complicated due to the central role of mitochondria in energy metabolism and redox homeostasis; apparently, the pathways relevant for cytosolic aggregate formation and mitochondrial functionality are intertwined in many ways. Yeast cells have been extensively used in the past as a rather simple and well-defined model system to unravel details of cytosolic homeostasis with a special focus on the role of mitochondrial protein import for the formation of cytosolic aggregates (Lu, Psakhye et al, 2014; Yang, Hao et al, 2016; Gruber et al, 2018; Weidberg & Amon, 2018). In order to study the role of mitochondrial protein biogenesis for cytosolic proteostasis, we expressed an established aggregating model protein in the baker's yeast Saccharomyces cerevisiae. The first exon of human huntingtin encodes an aggregation-prone poly-glutamine stretch (polyQ). Expression of polyQ fragments in yeast faithfully recapitulates huntingtin aggregation in a polyQ length-dependent manner (Braun, Buttner et al, 2010). We observed that the expression levels, mitochondrial localization, and functionality of Mia40 are critical determinants for polyQ toxicity. Mia40 (in humans also CHCHD4) is the rate-limiting essential factor of the machinery that imports proteins into the intermembrane space (IMS) of mitochondria (Chacinska, Pfannschmidt et al, 2004; Naoe, Ohwa et al, 2004; Peleh, Cordat et al, 2016; Peleh, Zannini et al, 2017; Habich, Salscheider et al, 2019). Increased levels of Mia40 in the IMS counteract the occurrence of aggregate-inducing nucleation seeds formed by the prion-like protein Rnq1 and suppress the growth arrest induced by an aggregation-prone polyQ protein. Overexpression of other components of the mitochondrial import machinery, in particular those relevant for the biogenesis of membrane proteins, was also able to suppress polyQ toxicity, pointing to a particular relevance of hydrophobic precursor proteins for cytosolic proteostasis. In general, our results show that the regulation of the mitochondrial import machinery, and in particular the modulation of the levels of Mia40, serves as an efficient molecular mechanism to finetune cytosolic protein homeostasis. Results Expression of Q97-GFP rapidly induces cytosolic aggregates and stalls cell growth The first exon of human huntingtin, comprising the polyQ stretch fused to green fluorescent protein (GFP), has been used in the past to study the formation of aggregates in the yeast cytosol (Krobitsch & Lindquist, 2000; Duennwald, Jagadish et al, 2006; Klaips, Gropp et al, 2020). We expressed a non-aggregating variant of 25 and an aggregation-prone variant of 97 glutamine residues fused to GFP (Q25-GFP and Q97-GFP, Fig 1A) from a regulatable GAL1 (for short here also referred as GAL) promoter in wild-type yeast cells. We initially cultured the cells in a galactose-free lactate medium to induce respiration and to stimulate mitochondrial biogenesis, before we induced the expression of the polyQ proteins by shifting cells to lactate medium that contained 2% galactose. Upon induction in galactose-containing medium, Q25-GFP expression resulted in a homogeneous cytosolic distribution, whereas Q97-GFP was predominantly forming aggregates seen as punctate signals (Fig 1B). Expression of Q97-GFP but not that of Q25-GFP was toxic and prevented cell growth (Fig 1C). Figure 1. The expression of Q97-GFP in the cytosol leads to the formation of insoluble protein aggregates Schematic representation of the structure of the Q25-GFP and Q97-GFP model proteins used in this study. The sequence consists of a FLAG-tag (turquois) followed by the polyQ domain from huntingtin exon1 (orange) and eGFP. Q25-GFP and Q97-GFP were expressed in wild-type cells for 18 h before cells were visualized by widefield fluorescence microscopy. Cells harboring an empty vector (ev) are shown for control. Cell boundaries are indicated by dashed lines. Bars, 5 µm. Wild-type cells expressing the indicated proteins under control of a galactose-inducible GAL1 promoter were grown in glucose medium to mid-log phase before tenfold serial dilutions were dropped on plates containing glucose or galactose. Wild-type (WT) cells were shifted from glucose to galactose. Then, mRNA levels of Q97-GFP were measured by qPCR. Shown are normalized mean values and standard deviations from three replicates. Cells were lysed with SDS-containing sample buffer before proteins were visualized by Western blotting. Insoluble aggregates migrate at the top of the gel between stacker and resolving gel. Tim17 served as loading control. Microscopic images of the Q97-GFP fluorescence. Bars, 5 µm. The percentage of cells containing or lacking detectable aggregates was quantified. Cell boundaries are indicated by dashed lines. Source data are available online for this figure. Source Data for Figure 1 [embj2021107913-sup-0009-SDataFig1.pdf] Download figure Download PowerPoint To analyze the order of events in polyQ-mediated toxicity, we analyzed cells after different times of induction. The Q97-GFP mRNA was detectable already after 10 min of induction and reached a maximum after about 90 min (Fig 1D). The Q97-GFP protein was well observed after 30 min and also reached a maximum after 90–120 min (Fig 1E). Interestingly, initially the Q97-GFP protein gave rise to a 47 kDa band on SDS gels closely matching the calculated molecular weight of the protein of 42.9 kDa. However, at later time points most of the protein was detected at the upper edge of the gel, indicative for the formation of SDS-insoluble protein aggregates (Douglas, Summers et al, 2009; Kim et al, 2016). The amounts of aggregated protein are presumably underestimated due to the limited solubility of this protein species. This aggregation behavior was confirmed by fluorescence microscopy where an initially homogeneous GFP signal was replaced by aggregates several hours after induction in an increasing number of cells (Fig 1F). After 4 h, about 50% of all cells that showed green fluorescence contained aggregates which increased to almost 100% after 6 h of expression (Fig 1G). Thus, the Q97-GFP protein is initially soluble and homogeneously distributed in the cytosol, but then rapidly aggregates in basically all cells, which coincides with a growth arrest. A functionally compromised mutant of the mitochondrial import factor Mia40 shows increased Q97 toxicity Previous studies suggested that cytosolic protein aggregation can disturb the functionality of mitochondria (Solans, Zambrano et al, 2006; Mossmann, Vogtle et al, 2014; Papsdorf, Kaiser et al, 2015). We therefore tested whether the Q97-GFP aggregates co-localize with mitochondria (Fig 2A). We observed that mitochondria and aggregates were clearly distinct structures. Mitochondrial co-localization that was seen for a few puncta might represent random contacts. Moreover, the formation of the Q97-GFP aggregates did not destroy the mitochondrial network which maintained its reticular structure for at least 4 h after induction of Q97-GFP expression (Fig 2A). We also monitored the respiration-driven oxygen consumption and the activity of cytochrome c oxidase which was only moderately reduced, even after 24 h of Q97-GFP expression (Fig 2B and C). We conclude that the expression of Q97-GFP induces the formation of cytosolic aggregates and impairs cell growth; however, at least within the first hours of induction, it only has mild effects on mitochondrial morphology and the functionality of the respiratory chain. Figure 2. Temperature-sensitive mia40-3 cells are hypersensitive to polyQ aggregates A. Mitochondria were visualized by mitochondrially targeted mCherry (red) in WT cells expressing Q97-GFP for 4 h. Fluorescence micrographs are maximum intensity projections of z stacks subjected to deconvolution. Bar, 5 µm. B, C. The polyQ proteins were expressed for the indicated time periods. Mitochondria were isolated from wild-type strains containing the Q25/97-GFP-expressing plasmids and from cells lacking mitochondrial DNA (rho0). The ability of these mitochondria to respire (shown as NADH-induced oxygen consumption) or their activity of cytochrome c oxidase (shown as their capacity to oxidize reduced cytochrome c) was measured. D. Cells of the temperature-sensitive mia40-3 mutant (Chacinska et al, 2004) or the corresponding wild type were transformed with plasmids expressing the indicated proteins from a low-expression GALL promoter (Mason et al, 2013) and grown on the respective carbon sources at permissive conditions (25°C). E. Q97-GFP was expressed in mia40-3 and corresponding wild-type cells for the times indicated. Cells were harvested and washed, and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown. Source data are available online for this figure. Source Data for Figure 2 [embj2021107913-sup-0010-SDataFig2.pdf] Download figure Download PowerPoint Next, we tested whether mitochondrial functionality influenced polyQ toxicity. Since the expression of the Q97-GFP from the GAL promoter was highly toxic, we used a plasmid which expressed Q103-GFP from a low-expression GALL promoter (Mason et al, 2013) and tested its effect on the growth in a temperature-sensitive Mia40 (mia40-3) mutant. Mia40 is an essential protein of the mitochondrial protein import machinery (Chacinska et al, 2004; Naoe et al, 2004), and the discovery that cytosolic precursors of mitochondrial proteins are toxic was initially made in such a temperature-sensitive Mia40 mutant (Wrobel et al, 2015). We observed that the mia40-3 mutant was more sensitive to Q103-GFP expression than the wild type: Even at permissive temperatures, cells were unable to grow on galactose-containing plates (Fig 2D). This suggests that the burden of non-imported mitochondrial precursors in the cytosol adds to the problems caused by toxic polyQ proteins. To test whether the effects on growth are due to a growth arrest or to cell death, we expressed high levels of Q97-GFP in wild-type and mia40-3 cells and grew them to log phase. Cells were exposed for 4 or 6 h to galactose, reisolated, and washed before surviving cells were counted by a plating assay on glucose medium. We observed that viability of Q97-GFP-expressing cells rapidly declined, in particular in the mia40-3 mutant (Fig 2E), suggesting that the growth arrest is caused by cell death (Chacinska, Lind et al, 2005). Overexpression of Mia40 suppresses the aggregation and toxicity of Q97-GFP The observed hypersensitivity of mia40-3 cells to Q97-GFP expression inspired us to test the effect of Mia40 overexpression. To this end, we made use of a strain which expresses Mia40 from a GAL promoter and which, in addition, harbors a plasmid with MIA40 under the control of its endogenous promoter (Terziyska, Grumbt et al, 2007). This extra-copy of MIA40 allows the strain to grow on glucose where the GAL-driven expression is repressed. This strain was transformed with the Q25-GFP and Q97-GFP expression plasmids. To our surprise, we observed that co-overexpression of Mia40 indeed strongly protected cells against the Q97-GFP-mediated growth arrest (Fig 3A). The Mia40-induced suppression was not due to reduced Q97-GFP expression as GAL-Mia40 cells even contained higher Q97-GFP levels than wild-type cells, albeit the levels of Q97-GFP were much lower than those of the non-toxic Q25-GFP variant (Fig 3B). Intriguingly, upon Mia40 overexpression Q97-GFP hardly formed any SDS-resistant aggregates, indicating that elevated levels of Mia40 suppressed aggregation of Q97-GFP. Figure 3. Overexpression of Mia40 suppresses polyQ toxicity in yeast Indicated strains were grown on glucose medium before tenfold serial dilutions were dropped on glucose- or galactose-containing plates. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; and ev, empty vector. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm. The patterns of the Q97-GFP distribution were quantified after different time of expression, n = 100. Cell boundaries are indicated by dashed lines. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown. The GAL-Mia40 strain was much more resistant to Q97-GFP expression than WT cells, indicating that high Mia40 levels can suppress polyQ toxicity. Source data are available online for this figure. Source Data for Figure 3 [embj2021107913-sup-0011-SDataFig3.pdf] Download figure Download PowerPoint Fluorescence microscopy confirmed the striking difference in aggregate formation (Fig 3C and D): Whereas Q97-GFP formed several small, distinct aggregates in wild-type cells, the protein was either homogeneously dispersed in the GAL-Mia40 overexpression strain or formed one large intracellular aggregate. Such large polyQ aggregates were described before in mutants of cytosolic chaperones (Krobitsch & Lindquist, 2000; Meriin, Zhang et al, 2002; Dehay & Bertolotti, 2006; Yang et al, 2016; Higgins, Kabbaj et al, 2018). Upon expression of Q25-GFP or Q97-GFP, mitochondria still formed a wild type-like reticulate structure, both in the presence and in the absence of Mia40 overexpression (Fig EV1). In some cells that simultaneously overexpressed Q97-GFP and Mia40, we noticed more reticulate, "curly" mitochondria (Fig EV1). These structures were not observed when only one of these proteins was overexpressed. We are not aware that such structures were reported before, and their significance is currently unclear. Click here to expand this figure. Figure EV1. Co-expression of Q97-GFP and GAL-Mia40 results in minor changes of mitochondrial morphology Cells were grown to mid-log phase in glucose-containing medium, shifted to galactose-containing medium for 4 h, and analyzed by 3D fluorescence microscopy. Fluorescence micrographs are z stacks subjected to deconvolution. DIC, differential interference microscopy. Bar, 5 µm. Cells were grown to mid-log phase in medium containing glycerol (3%) and ethanol (2%) as carbon sources, shifted to galactose-containing medium for 4 h, and analyzed by 3D fluorescence microscopy. Fluorescence micrographs are maximum intensity projections of z stacks. Asterisks indicate representative cells exhibiting interconnected, "curly" mitochondria. Bar, 5 µm. Download figure Download PowerPoint Mia40 overexpression was very effective in the repression of polyQ toxicity and about half of all cells even survived the GAL-driven expression of Q97-GFP for 24 h (Fig 3E). From this, we conclude that the overexpression of the mitochondrial import factor Mia40 can protect against the formation of toxic polyQ aggregates (Fig 3F). Mia40 influences the formation and inheritance of polyQ aggregates Since we had observed that induction of Q97-GFP in wild-type cells resulted in an initially homogeneously distributed protein that later formed SDS-resistant aggregates, we wanted to better understand the temporal and spatial patterns of aggregate formation and inheritance in wild-type and GAL-Mia40 cells. To this end, we monitored the growth of wild-type and GAL-Mia40 cells after galactose-driven induction of Q25-GFP and Q97-GFP in a microfluidics growth chamber (Morlot, Song et al, 2019) under the fluorescence microscope (Fig 4A). Thereby, individual cells could be followed over time which showed that GAL-Q25 levels did not interfere with bud formation and growth. In contrast, in wild-type cells Q97-GFP expression slowed down cell division, and cells died prematurely (Fig 4B, Movies EV1–EV3). The pattern was entirely different in GAL-Mia40 cells: Cells accumulated Q97-GFP with a homogeneous intracellular distribution and thereby reached higher fluorescence levels than wild-type cells (Fig 4B and C). However, we noticed that, i
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