bioRxiv preprint doi: https://doi.org/10.1101/253153; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Analysis of heat-induced protein aggregation in human mitochondria Anne Wilkening, Cornelia Rüb, Marc Sylvester and Wolfgang Voos* From the Institute for Biochemistry and Molecular Biology, Friedrich-Wilhelm-University of Bonn, Germany Running title: Protein aggregation in heat-stressed mitochondria *To whom correspondence should be addressed: Wolfgang Voos: Institut für Biochemie und Molekularbiologie, Nussallee 11, 53115 Bonn, Germany; [email protected]; Tel. +49-228-732426; Fax: +49-228-735501 Keywords: Mitochondria, heat-stress, proteome analysis, protein aggregation, protein translation, mitochondrial elongation factor Tu (Tufm), preprotein import Abstract Introduction As proteins in mammalian cells exhibit Mitochondria are organelles that fulfill optimal stability at natural temperatures, many essential cellular tasks in addition to small temperature variations may cause their traditionally established functions in unfolding and subsequent non-specific energy metabolism. These functions are aggregation. As this process leads to a dependent on the enzymatic activities loss of function of the affected provided by many different mitochondrial polypeptides as well as to further cytotoxic polypeptides, of which the majority is stress, aggregate formation has been encoded in the nucleus while only a few recognized as a major pathogenic factor in polypeptides are generated by the human diseases. In this study we endogenous mitochondrial protein bio- determined the impact of physiological synthesis machinery. The mitochondrial heat stress on mammalian mitochondria fitness is therefore dependent on a on a proteomic level. The overall solubility coordinated expression of both nuclear of endogenous mitochondrial proteins was and mitochondrial genes. The only marginally affected by a treatment at mechanisms underlying this coordination elevated temperatures. However, we process, in particular during adaptation to identified a small subset of polypeptides various stress conditions, are still only that exhibited an exceptionally high inadequately known and are an active field sensitivity to heat stress. The of cell biological research (1). mitochondrial translation elongation factor To maintain mitochondrial functions Tu (Tufm), a protein essential for under normal and stress conditions, a organellar protein biosynthesis, was highly mitochondria-specific protein quality aggregation-prone and lost its solubility control (PQC) system, consisting of already under mild heat stress conditions. chaperones and proteolytic enzymes, In parallel, mitochondrial translation as protects the structural integrity of well as the import of cytosolic proteins was endogenous polypeptides or removes defective in heat stressed mitochondria. terminally misfolded proteins (2). Apart Both types of nascent polypeptides, from a loss of important enzymatic derived from translation as well as from activities, an accumulation of misfolded import exhibited a strong heat-induced proteins can also lead to aggregation aggregation tendency. We propose a reactions that result in further structural model that a quick and specific inactivation and functional damage of the cells. As of elongation factors may prevent an shown in other organelles or in the cytosol, accumulation of misfolded nascent the activity of the PQC system is mainly polypeptides and thereby attenuate aimed at the prevention of an proteotoxicity under stress. accumulation of cellular protein 1 bioRxiv preprint doi: https://doi.org/10.1101/253153; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. aggregates (3). The proteotoxic effects of of molecular chaperones during stress aggregates are best exemplified in the (13). If this protein quality control system is occurrence of so-called protein-folding overwhelmed, cells have additional diseases, in particular neurodegenerative protective mechanisms to deal with diseases that are caused by or at least aggregated proteins, like sequestration to correlated with the accumulation of specific compartments, disaggregation specific protein aggregates (4). The and refolding to a native protein structure, chronic expression of aggregation–prone clearance by autophagy as well as proteins such as pathological poly- asymmetric distribution of aggregates glutamine proteins in these diseases during cell division (9,14). declines the protein homeostasis of the Although substantial information on the cell, which results in further misfolding and stress-related reactivity of the cytosolic aggregation of metastable proteins. compartment has been obtained, so far Further the overall capacity of protein there is only little known about the homeostasis is declined in aging cells, aggregation behavior of endogenous which results in an increased risk of mitochondrial proteins under stress accumulation of toxic protein aggregates conditions and the effect on the overall (5,6). Research efforts over the past mitochondrial function. Previous studies in decade have established that mito- S. cerevisiae have shown that key chondrial dysfunction plays a major role in mitochondrial metabolic enzymes are the etiology of many neurodegenerative aggregating and become inactivated under diseases (7) as well as in aging processes elevated temperatures. The aggregation (8). propensity was modulated by Besides mutations and translation mitochondrial chaperones and the Pim1 errors, there are also environmental stress protease in the matrix compartment (15). conditions, which cause protein misfolding Interestingly, mitochondrial proteins were and consequently lead to aggregation over-represented in a screen of aging- processes (9). Elevated temperatures or related protein aggregation in C. elegans, heat stress at non-lethal levels represent a connecting mitochondrial protein prominent cause of polypeptide aggregation with age-related diseases denaturation and functional inactivation. (16). As already small increases in temperature In this work, we studied the impact of can lead to protein unfolding and mitochondrial protein aggregation aggregation, elevated growth reactions at different stress conditions temperatures represent a substantial under in vivo like conditions in challenge to cellular survival, mitochondria isolated from mammalian characterized by a damaged cytoskeleton, cells. We identified aggregation–prone fragmentation of organelles, swelling of mitochondrial proteins by performing a the nucleus, formation of stress granules quantitative proteomic analysis and arrest of the cell cycle (10). However, determining the changes in protein a small increase of the physiological solubility after heat stress. In particular, we temperature could also be beneficial for subjected isolated intact mitochondria to a organisms under specific circumstances, short heat shock treatment, representing as fever in vertebrates has a survival physiological heat stress conditions, to benefit by regulating the immune system induce protein aggregation and and protecting the organism against characterize the changes in protein infection (11). abundance using 2-dimensional Elevated temperatures activate a differential gel electrophoresis (2D-DIGE). cellular heat shock response by inducing The quantification of the protein spot the synthesis of heat shock proteins that pattern allowed an identification of help to maintain protein homeostasis and temperature-sensitive polypeptides, whose ensure the survival of the cell (12). The aggregation behavior was further heat shock response is based on a characterized in more detail. Finally, we specific transcriptional response regulated tested the impact of heat stress on the by heat shock transcription factors (HSF) mitochondrial protein biogenesis reactions that induce the synthesis of a specific set by assessing mitochondrial translation as 2 bioRxiv preprint doi: https://doi.org/10.1101/253153; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. well as the efficiency of polypeptide interested if there is a change in the ATP import. levels in isolated mitochondria after incubation at elevated temperatures. Our Results results showed that there was already a decrease of about 25% in the General effects of heat stress on cells mitochondrial ATP content after 20 min and isolated mitochondria. incubation at 25°C, which was increased To determine the effects of heat stress after heat shock, resulting in a decrease of on the functional integrity of mitochondria, more than 80% compared to pre-heat we measured typical metabolic properties shock levels (Fig. 1D). of mitochondria, like the inner membrane As heat-treated cells exhibited a potential (Δψ), the accumulation of significant change of the mitochondrial reactive oxygen species (ROS), and the morphology as investigated by mitochondrial ATP levels. The membrane fluorescence microscopy (Fig. S1), potential of HeLa cells was analyzed by changing the typical filamentous network staining with the potential-dependent structure to a more condensed structure mitochondrial dye tetramethylrhodamine