Thermal Shock Induces Host Proteostasis Disruption and

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Thermal shock induces host proteostasis disruption and endoplasmic reticulum stress in the model symbiotic cnidarian Aiptasia Clinton Alexander Oakley, Elysanne Durand, Shaun P. Wilkinson, Lifeng Peng, Virginia M. Weis, Arthur R. Grossman, and Simon K. Davy J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017

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Journal of Proteome Research

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Thermal shock induces host proteostasis disruption and endoplasmic reticulum stress in the model symbiotic cnidarian Aiptasia Clinton A. Oakley*1, Elysanne Durand2, Shaun P. Wilkinson1, Lifeng Peng1, Virginia M. Weis3, Arthur R. Grossman4, and Simon K. Davy1. 1

School of Biological Sciences, Victoria University of Wellington, Wellington, 6012, New Zealand 2 Department of Ecology and Environmental Sciences, Université Pierre et Marie Curie, Paris, 75005, France 3 Department of Integrative Biology, Oregon State University, Corvallis, Oregon, 97331, United States 4 Department of Plant Biology, The Carnegie Institution for Science, Stanford, California, 94305, United States

*Correspondence: Clinton A. Oakley, School of Biological Sciences, Victoria University of Wellington, Wellington, 6012, New Zealand. +64 04 4639802 [email protected]

Running title: Thermal proteostasis disruption in the model Aiptasia

Abstract

ACS Paragon Plus Environment


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Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 °C and 33.5 °C, 12 d) elevated temperatures on the proteome of the model symbiotic anemone Aiptasia. We identified 2,133 proteins in Aiptasia, 136 of which were differentially abundant between treatments. Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and central metabolism. Nineteen abundant structural proteins showed particularly reduced abundance, demonstrating proteostasis disruption and potential protein synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing protein aggregation, and degrading damaged proteins, indicative of endoplasmic reticulum stress. Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting hostderived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes are essential in the cellular response of symbiotic cnidarians to severe thermal stress. Key words: Aiptasia, Cnidaria, coral reefs, endoplasmic reticulum, label-free proteomics, symbiosis, thermal stress, unfolded protein response


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Introduction Corals are the engineers of highly diverse coral reef ecosystems, building the physical structure of the reef and contributing to reef nutrient cycling through their production of photosynthates and labile mucus1. Reef-building corals are a mutualism between the coral animal host and intracellular dinoflagellate symbionts of the genus Symbiodinium, in which algal photosynthates are exchanged for nitrogenous compounds and inorganic carbon from the host2. Existing in a highly energetic environment in shallow tropical seas and exposed to warm temperatures and high ultraviolet radiation, coral reefs require a narrow temperature range and are sensitive to extreme thermal events3. These abiotic stresses elicit damage DNA, membranes, and proteins, and induce universal cellular stress responses (CSRs)4,5. Corals are incapable of thermal homeostasis and, as sessile organisms, must acclimate in situ at the cellular level to changes in water temperature. The coral–dinoflagellate symbiosis and reefs worldwide are increasingly threatened by anthropogenic climate change, ocean acidification, and coral bleaching, in which periods of high sea surface temperatures result in the loss of algal symbionts from the host, potentially resulting in regional coral mortality6,7. Coral tissues possess ideal conditions for the generation of reactive oxygen species (ROS) owing to their high metabolic rates and the abundant oxygen produced by photosynthesis of their endosymbiotic algal partners8. There have been extensive efforts to investigate the roles of light, ROS and oxidative damage during thermal stress in symbiotic cnidarians3,9–12, and algal symbiont-derived ROS have been proposed as instigators of the bleaching response8,12. Recently, increased attention has been paid to the role of host endogenous ROS and nitric oxide (NO) generation during cnidarian thermal stress10,13. The endoplasmic reticulum (ER) is a potent source of up to 25% of total ROS generation in eukaryotic cells due to the transfer of electrons to



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O2 during peptide disulfide bond formation14. Maintaining proteome stability, or proteostasis, during thermal stress depends on the rapid initiation of the highly conserved heat shock response, primarily the induction of heat shock proteins (Hsps), which serve as chaperones to maintain correct protein folding15. Misfolded proteins are potentially toxic and prone to aggregation, and must be repaired or destroyed15. The unfolded protein response (UPR) is induced by the aggregation and import of misfolded proteins beyond the processing capacity of the ER proteinfolding machinery, and it mitigates ER stress by re-folding misfolded or damaged proteins16,17. ER stress provokes a number of cellular responses in addition to the UPR, including ER expansion, increased phospholipid synthesis, chaperone upregulation, reduced global protein translation, and enhanced protein export from the ER16. Collectively, these mechanisms serve to increase protein-folding capacity and purge misfolded or aggregated proteins from the ER18. If these mechanisms are insufficient, the terminal step of the UPR is apoptosis. Host apoptosis has been demonstrated in response to thermal stress in symbiotic cnidarians19, but its role in bleaching is disputed20. Determining the mechanisms used by the cnidarian host to acclimate to varying thermal conditions and severe thermal stress is important to better understand the resilience of the coral– dinoflagellate mutualism in future climate-change scenarios. Corals are differentially susceptible to bleaching due to host and symbiont identities, local physical oceanography (e.g. depth and water flow), and frequency of anomalous thermal events21. Thermal coral bleaching can be induced by sea surface temperature fluctuations of as little as 1–2 °C greater than the average maximum sustained temperature22. Therefore, the host physiological mechanisms of acclimation to high temperatures, as well as the CSRs induced by rapid elevation in temperature (“heat shock”), are crucial. The circumtropical anemone Aiptasia is a model organism for studies of the


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coral–dinoflagellate symbiosis23,24, and genetic and gene expression databases of this system are rapidly expanding25–27. To compare the effects on the cnidarian host of gradual high-temperature acclimation to those of a rapid thermal assault, Aiptasia were either acclimated to higher temperatures over a period of two weeks or were exposed to short-term (24 h) thermal shock without acclimation. We then used label-free liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) “shotgun” proteomics to characterize the Aiptasia proteome following each of these treatments. We performed a detailed analysis of the specific proteins and cell mechanisms induced by acclimation and heat shock, providing additional insight into the cellular biology of the initial phases of coral thermal stress and the interplay between the host and symbiont prior to detectable bleaching.

Materials and Methods Aiptasia culture and physiology Aiptasia sp. (= Exaiptasia pallida28) were isolated from a Pacific-sourced aquarium and a clonal population, strain NZ1, was maintained in the laboratory for several years at 25 °C and approximately 40 µmol quanta m−2 s−1. We have maintained using the Aiptasia nomenclature here for agreement with published genomic resources26. This host strain associates with Symbiodinium minutum (clade B)24,29. Pairs of anemones were maintained in 500-mL glass beakers at 25 °C and 100 µmol quanta m−2 s−1 with a 12 h:12 h light:dark cycle. Anemones were allowed to acclimate to these conditions for two weeks. Anemones were fed Artemia sp. nauplii and the water was changed with 0.22 µm-filtered seawater twice per week. To minimize contamination from Artemia protein, anemones were not fed for one week prior to sampling. The dark-adapted quantum yield of chlorophyll fluorescence of 3–6 anemones per treatment was



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measured daily using a diving pulse-amplitude modulated (PAM) fluorometer (Heinz Walz GmbH, Effeltrich, Germany) to monitor the maximum efficiency of photosystem II, beginning four days before the start of the experiment. Sampling was conducted by homogenizing the pooled anemones, followed by separating the algal symbionts from the host homogenate by centrifugation. Symbionts were fixed with formalin and counted using a hemocytometer to assess symbiont density. Sample protein content was determined by the Bradford assay. All reagents were acquired from Sigma-Aldrich (Auckland, New Zealand) unless noted. Experimental design and statistical rationale Anemones were exposed to four thermal treatments: control (25 °C), 30 °C with acclimation, 33.5 °C with acclimation, and a 33.5 °C “heat shock” treatment without thermal acclimation. These temperatures were chosen to represent the range known to induce sub-lethal thermal stress and bleaching in Aiptasia13,30. Six biological samples (n = 6), each consisting of two pooled anemones, were analyzed per treatment, and each biological sample (24 total) was analyzed twice, forming technical replicates. After two weeks of acclimation under control conditions, the temperature of the anemones in the 30 °C and 33.5 °C-acclimated treatments was increased by 1 °C per day to 30 °C, and the anemones were maintained at this temperature for five days. For the 33.5 °C-acclimated treatment, the temperature was further increased to 33.5 °C over seven days, followed by sampling. Control, 30 °C-acclimated, and 33.5 °C-acclimated anemones were sampled simultaneously. Anemones exposed to heat shock were acclimated to the 25 °C control tank for two weeks, after which the temperature was increased to 33.5 °C over 1 h, and the anemones were sampled 24 h later. All samples were analyzed in a stratified random order to minimize the effects of instrument drift. LC-ESI-MS/MS methods


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Samples (20 µg final protein mass) were prepared by a sodium deoxycholate in-solution digestion method with 1 µg of sequencing-grade trypsin using the methods and instrument settings described in Oakley et al.27. Samples were analyzed by LC-ESI-MS/MS with a 300 min gradient (buffer A: 0.1% formic acid; buffer B: 80% acetonitrile, 0.1% formic acid) at 0.3 µL min−1 on an Acclaim PepMap C18, 3 µm, 100 Å column (Thermo Scientific, Auckland, New Zealand) and Ultimate 3000 (Dionex, Sunnyvale, California, USA), with the column oven at 35 °C. An LTQ Orbitrap XL (Thermo Scientific) was used to analyze peptides that were injected at a 1.9 kV spray voltage with a resolution of 30,000. The ion trap analyzed MS/MS spectra from the top eight MS peaks, rejecting +1 charge states with dynamic exclusion enabled (180 s). The LC-ESI-MS/MS instruments were operated with Chromeleon Xpress (v2.11.0.2914, Dionex), Thermo Xcalibur (v2.1), and ThermoTune Plus (v2.5.5, Thermo Scientific). Spectra were searched using a custom sequence database previously used for Aiptasia protein identification27. This database consists of the Aiptasia genome26, all UniProt cnidarian sequences, all open reading frames from an Aiptasia strain CC7 transcriptome25, and a database of common contaminants31 (320,798 total sequences) and was processed by MScDb to reduce peptide-level redundancy32. Peak lists were generated by Proteome Discoverer (v1.4.1.14, Thermo Scientific) via SEQUEST HT searches33 followed by Percolator scoring. SEQUEST HT searches assumed trypsin digestion with a maximum of two missed cleavages, one minimum matching peptide per protein, a minimum peptide length of six amino acids, a maximum delta Cn of 0, a parent ion tolerance of 10.0 parts per million, and a fragment ion mass tolerance of 0.60 Da. Carbamylation of the N-terminus and oxidation of methionine were specified as variable modifications and carbamidomethylation of cysteine was specified as a fixed modification.



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Proteins were quantified by total ion current (TIC) in Scaffold (Table S1), and statistical analyses were carried out in R34. The TIC values for all protein clusters per sample were summed to determine if TIC normalization between samples was necessary. Variation in grand total TIC values was much greater between biological replicates than between technical replicates, indicating differences in total protein loading between samples (Fig. S1). To account for differences in protein loading between samples, a scaling factor approach was used based on the assumption that the majority of proteins are not differentially expressed in response to changes in temperature or by indirect thermal effects (e.g. different growth rates). A pairwise matrix of scaling factors for each sample (x) against each other sample (y) was calculated as the median of the vector = 1, 2, 3, … , where m is the total number of proteins that were detected in all 48 samples (345 proteins). The sample with the lowest protein loading depth (the reference sample) was identified as the matrix column with the lowest average scaling factor. All other samples were then normalized to this sample using the scale factors from the reference sample column. To aid in parametric model-fitting, TIC values were then assigned for low-abundance protein clusters whose concentrations were below the detection limit of the instrument (“nondetects”). Protein clusters with non-detect rates exceeding 33% were removed from the dataset, and the non-detects in the remaining clusters were imputed by robust regression on order statistics (assuming a log-normal distribution of protein concentrations within samples) using the R package ”NADA”35. Following imputation, the technical replicates were averaged, and protein clusters that differed in concentration between treatments were identified. For multivariate analysis of treatment effects, the dataset was log-transformed, and Bray-Curtis dissimilarities


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were calculated for each pairwise sample comparison. Analysis of variance by permutation of dissimilarities was carried out using the “adonis” function in the R package “vegan” (v2.2-1)36. To test for differences in individual protein clusters between treatments, a generalized linear model with treatment effects was fitted and compared against a null model (intercept only) using an analysis of deviance Χ2 test with α = 0.05. Protein clusters showing differences in concentration between treatments were identified with the false discovery rate (FDR) correction procedure of Benjamini & Hochberg37, using a q-threshold of 0.1 (Table S2). To ensure that the regression on order statistics imputation procedure did not have an undue impact on the results, the procedure was repeated 100 times with random reallocations of imputed values. Scaffold (v4.3.4, Proteome Software Inc.) was used for further validation and for quantification via label-free TIC using the SEQUEST HT and X!Tandem algorithms (The GPM, thegpm.org; version Cyclone 2010.12.01.1). Protein probabilities were assigned by Protein Prophet38. FDRs were calculated using a decoy database39. The FDR threshold for reported peptides and proteins was set to 0.1% and 1%, respectively, with a minimum of two peptides per protein. Proteins that shared significant peptide evidence were grouped into clusters using Scaffold’s Protein Cluster Analysis algorithm. Following further statistical analysis (see above), protein clusters that were found to be significantly different between treatments were annotated by manual BLAST searches of each database sequence against the UniProtKB database. Each protein cluster was given the annotation of the UniProtKB entry with the highest BLAST score that had an E-value of less than 1.0 × 10−10. Functions were inferred from these annotations, and fold changes (FC) were derived by performing log2 transformation of protein abundance ratios. To further verify the functions of significantly different proteins, UniProtKB entries were assigned biological process gene ontology (GO) terms using QuickGO40,41. Mass spectrometry



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data have been deposited to the ProteomeXchange Consortium42 via the PRIDE partner repository with the dataset identifier PXD004257.

Results and Discussion Symbiont density and Fv/Fm are widely-used physiological metrics of coral bleaching43, and our data indicate that changes in the host proteome preceded the breakdown of the symbiosis. Clear signs of thermal stress were observed in the host proteome, however, symbiont density was not significantly different at the time of sampling among any of the treatments (Fig. 1A, p > 0.05, single-factor ANOVA). Furthermore, none of the treatments resulted in a significant change in the dark-adapted quantum yield of photosystem II (Fv/Fm), a measure of the photophysiological health of the dinoflagellate symbionts44 (Fig. 1B).


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Figure 1. Density and quantum yield of photosystem II chlorophyll fluorescence of algal symbionts in Aiptasia. Anemones were kept at 25 °C (25 °C), acclimated to 30 °C (30 °C), acclimated to 33.5 °C (33.5 °C), or heat-shocked at 33.5 °C for 24 h without thermal acclimation (33.5 °C HS). A) Algal cell density at the time of proteome sampling, n = 6. B) Dark-adapted quantum yield of photosystem II chlorophyll fluorescence of Aiptasia symbionts prior to and during the thermal treatment, n = 3–6 per time point. Day 0 (dashed line) indicates the end of the acclimation period and the beginning of the thermal treatments.



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The levels of many highly abundant host proteins were greatly reduced following heat shock conditions (33.5 °C, 24 h) when compared with those corresponding proteins in control (25 °C) or acclimated (30 °C and 33.5 °C) animals (Table 1). These proteins include several components involved in motor function, cellular matrix structure, and protein trafficking. We identified a total of 2,187 protein clusters (from here on referred to as “proteins”) across all treatment groups using X!Tandem (Table S2). The 25 proteins with the greatest average TIC values are listed in Table 1 in order of their average abundance across all samples, as determined by the percentage of the individual protein TIC values relative to that of all proteins in the given sample. We discarded 45 decoy sequences and nine contaminant proteins from the dataset while 1,253 proteins were not analyzed further due to having more than 33% nondetects. Of the remaining 934 proteins, 593 had at least one value imputed. For all 100 iterations, the multivariate p-values for all differentially-expressed protein clusters identified in the generalized linear model analysis were less than 0.05. Significant differences existed between treatments (multivariate p-value = 0.002). After anemones were acclimated to 30 °C or 33.5 °C, there were only eight and nine proteins, respectively, that were differentially abundant (q < 0.1) between the control and acclimated animals (Tables 2, 3). In contrast, there were many more differentially abundant proteins between the heat shock treatment and the control, 30 °C acclimation, and 33.5 °C acclimation treatments (104, 49, and 74 proteins, respectively; Table 2). The proteins that had significantly different abundances between the control and heat shock treatments are given in Table 4, where they are organized by biological function. Differentially abundant proteins between the control and heat shock treatments were also assigned biological process GO terms (Fig. 2). A complete list of differentially abundant proteins is given in Table S3.


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Figure 2. Counts of proteins that were differentially abundant (q < 0.1) between control (25 °C) and heat-shocked (33.5 °C, 24 h) Aiptasia, grouped by biological process gene ontology terms. A) More abundant proteins in heat-shocked Aiptasia (minimum four shown) or B) less abundant proteins in heat-shocked anemones (minimum three shown).

Only nine proteins were differentially abundant between control anemones kept at 25 °C and those that were allowed to slowly acclimate to 33.5 °C (Table 3). Of this limited set,



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metabolic enzymes include an increase in 75-kDa NADH-ubiquinone oxidoreductase subunit 1 (NDUFS1) and decreases in pyruvate carboxylase and phosphoinositide phospholipase Cη2. This minimal number of changes, in contrast with the larger differences between control and heat-shocked anemones, reflects the ability of the animal to maintain homeostasis through means that do not involve changes in protein abundance. Two elongation factors, EF1γ and EF2, exhibited reduced accumulation in anemones acclimated to 33.5 °C, which may indicate the arrest of cell division and animal growth under sustained elevated temperature45. Compared with their levels in control anemones, proteins that play roles in multiple thermal stress response mechanisms were upregulated in heat-shocked Aiptasia (Fig. 2). From here on, unless otherwise stated, “differentially abundant proteins” refer to changes between control (25 °C) and 33.5 °C heat-shocked treatments. Our proposed model (Fig. 3) interprets these changes as evidence of broad oxidative damage to proteins and disruption of proteostasis through synergistic oxidative and ER stress, resulting in an increase in proteins associated with redox control, protein synthesis, and protein degradation.


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Figure 3. Cellular processes proposed to be induced by thermal shock in Aiptasia. A) Cytoskeletal proteins are damaged by reactive oxygen species (ROS) exposure (red regions). The damaged proteins are ubiquinated (green circles) and targeted to the proteasome for degradation. The folding of new cytoskeleton proteins is stabilized by chaperonins (CCT). B) Nascent proteins either fold correctly (blue) or are misfolded (red). Misfolding rates increase under high temperatures, resulting in an accumulation of misfolded proteins and the induction of the unfolded protein response. C) Misfolded proteins are identified by heat shock proteins (Hsps), such as Hsp90, and are either repaired or unfolded for retrotranslocation from the endoplasmic reticulum (ER), which is then followed by ubiquination and subsequent degradation by the proteasome (ER-associated degradation, ERAD). Protein unfolding by protein disulfide



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isomerase results in ROS generation. D) Increases in coatomer (COPI) protein reflect an increased amount of COPI vesicle cycling between the ER and Golgi apparatus, which increases the influx of damaged or misfolded proteins into the ER. E) Proteins that are unable to be refolded are exported from the ER, ubiquinated and transported to the proteasome for degradation via ERAD. F) The secondary messenger Ca2+ is exchanged between the ER and mitochondria through tight junctions stabilized by HspA9. Transfer of Ca2+ from the ER to the mitochondria increases during ER stress, stimulating mitochondrial respiration and potentially inducing apoptosis. G) Upregulation of both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation increases the reductant available for antioxidant mechanisms as well as the generation by NADH-ubiquinone oxidoreductase (NDUFS1) of ROS, some of which may leak from the mitochondria. H) The antioxidant glutathione (GSH) repairs oxidized proteins, and its oxidized form (GSSG) is reduced by NADPH generated by the pentose phosphate pathway (PPP). GSH is synthesized as a result of the methionine/S-adenosyl methionine (SAMe) cycle, enzymes of which are upregulated by increased temperatures.

Structural and cytoskeletal proteins The effects of heat shock were most pronounced on the highly abundant cytoskeletal, actin-interacting, cell–cell binding and muscle fiber proteins, which together comprised 45% of the total identified protein biomass (Table 1, Fig. 3A). Nineteen proteins with structural, muscular, or cytoskeletal roles exhibited significantly lower abundances under heat-shock conditions compared with controls (Table 4), including 14 of the 25 most-abundant proteins. Interestingly, this group included the most abundant detected protein, β-actin, which exhibited a −1.1 log2 FC. Actin is the most abundant protein in the eukaryotic cytoskeleton and the principle


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component of the microfilament network; it is also a muscle fiber constituent and sensitive to oxidative damage46. Oxidative stress has been demonstrated to have broad effects on cytoskeleton organization and structure, as well as on the polymerization of its constituent proteins47. Collectively, cytoskeletal and structural protein abundances were reduced by 51% in heat-shocked anemones compared with control organisms. The large, concerted reductions in these proteins signify re-organization of the cell and disruption of multiple proteostasis mechanisms. Induction of the heat shock and unfolded protein responses The heat shock response and UPR are highly conserved mechanisms of responding to thermal disruption. Out of the 44 conservatively-defined “minimal stress proteome” (MSP) proteins that are universal among all three domains of life5, we detected ten with an increased abundance in heat-shocked Aiptasia compared with controls (Table 4). This group includes multiple classes of Hsps (Fig. 3B). Two Hsp70s were increased in heat-shocked anemones: the binding immunoglobulin protein (BiP; 1.2 FC) and stress-70 protein (HspA9; 2.8 FC). BiP is a principal ATP-dependent protein-folding and re-folding chaperone of the heat shock response localized in the ER48. HspA9 is an Hsp70-family chaperone located in the mitochondrial matrix that serves as both a chaperone and a regulator of the translocation of newly-synthesized proteins into the matrix17; critically, HspA9 upregulation moderates ROS damage, increases cell survival, and links the oxidative states of the mitochondria and ER. Similarly, two members of the Hsp90 family, Hsp90A and Hsp90B, were elevated after heat shock in different cellular compartments (1.1 FC/cytosol and 1.1 FC/ER, respectively). Both Hsp90s were highly abundant in all treatments, as is typical for this class of chaperone, which exhibits specificity for particular client proteins, particularly those involved in the protein secretory pathway49. Hsp60, another class of



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mitochondrial chaperone that also increased under heat shock (0.6 FC), is crucial for transmembrane trafficking of mitochondrial proteins50. The 1.8-FC increase of prohibitin-2, a mitochondrial chaperone responsive to oxidative stress that acts as a holdase/unfoldase (analogous to the ER Hsps), provides further evidence of a mitochondrial response to heat shock51. Maintaining optimal rates of protein folding, degradation and export from the ER is essential, and these processes are highly regulated (Fig. 3B–E). Heat-shocked anemones experienced proteostasis disruption and a need for rapid protein degradation and turnover, based on the rapidly increased abundances of many proteins involved in these mechanisms. Misfolded proteins are recognized by chaperones, including BiP and protein disulfide isomerases (PDIs), and targeted for translocation from the ER (Fig. 3C). PDIs are unusual chaperones in that they link the cellular redox state to proteostasis by being active in their reduced form, allowing them to unfold exogenous toxins and misfolded endogenous proteins52,53. Transport to and from the ER is accomplished via COPI vesicles lined with coatomer protein, which are involved both in the typical transport of proteins from the ER (Fig. 3D), as well as in the trafficking of misfolded or aggregated proteins back to the ER54. The levels of coatomer protein and two distinct PDIs were both greatly and similarly elevated in heat-shocked Aiptasia (2.8, 2.7, and 1.0 FC, respectively). Two highly abundant golgins, which play a role in inter-Golgi transport by COPI vesicles, were found to decline following heat shock (Tables 1, 3). Protein glycosylation is necessary for the correct folding of as many as 70% of the proteins that exit the ER55, and a required subunit of the rough ER protein oligosaccharyl transferase complex (OST1)54 was elevated (1.2 FC, Table 4).


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Proteins that are unable to be re-folded are directed to the ER-associated degradation (ERAD) system18 (Fig. 3E). ERAD works in concert with the UPR to reduce ER stress by facilitating ubiquitination of the misfolded proteins, which are then degraded by the 26S proteosome. The ubiquitin-proteasome system is responsive to oxidative stress and is responsible for the majority (>70%) of protein degradation in eukaryotes56–58. Transitional ER ATPase (VCP, 1.0 FC), a protein of the ERAD and ubiquitin systems, binds to polyubiquinated proteins and uses ATP to unfold or remodel targeted proteins, often facilitating their degradation by the proteasome59. We also detected a 2.3-FC increase of the proteasome 19S cap protein subunit Rpn1, which serves to shuttle bound ubiquinated proteins into the proteasome complex for degradation60. Together, the changes in the abundance of these proteins in heat-shocked anemones support a role for ERAD in maintaining proteostasis after heat shock in cnidarians. Given the dramatic reductions in cytoskeletal protein abundance after heat shock, largescale protein degradation and turnover would demand simultaneous increases in chaperones to assist in replacing the lost proteins. Chaperonins, a subset class of chaperones, are ring-shaped protein complexes that assist in protein folding by providing isolation and protection from the cytosolic milieu61. The cytosolic chaperonin containing TCP-1 (CCT) is a complex comprised of two eight-subunit rings62,63 (Fig. 3A), and we observed 1.0- to 1.6-FC increases of four CCT subunits in response to heat shock (Table 4). Up to 15% of all cytosolic proteins have been proposed to pass through CCT during folding and assembly64. The most important role of CCT is in the folding of cytoskeletal proteins like actin and tubulin65, which fold particularly slowly, on the order of several minutes, and are therefore susceptible to aggregation66. The induction of CCT and Hsps indicates a need for the re-synthesis of compromised proteins, particularly the highly abundant cytoskeletal components, after proteome disruption.



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Calcium-binding proteins Calcium is an important secondary messenger ion, and changes in Ca2+ concentration induce many CSRs67. The ER substantially envelops mitochondria, and Ca2+ communication between the two occurs through multiple mechanisms, including direct exchange through tight junctions stabilized by the cytosolic isoform of HspA917,67 (Fig. 3F). Ca2+ transfer to the mitochondria stimulates respiratory and TCA cycle activity, eventually inducing apoptosis,17 and the role of Ca2+ homeostasis in cnidarian bleaching has been previously explored68–70. Our results detail large abundance shifts in cytoskeletal proteins, many of which have Ca2+-binding activity, as well as in specific Ca2+ regulators. Calumenin is a Ca2+-binding EF-hand-containing protein that is localized to the ER. It has been demonstrated to be upregulated in response to ER stressors, including oxidative stress, and plays a chaperone-like role in alleviating ER stress and subsequent apoptosis10,71. Calumenin was found to be greatly upregulated in heat-shocked Aiptasia (4.5 FC). Ganot et al. suggested a role of calumenin in the cnidarian-dinoflagellate symbiosis, demonstrating multiple cnidarian-specific gene duplications and strong upregulation of calumenin in the symbiotic sea anemone Anemonia viridis relative to aposymbiotic animals72. Bellantuono et al. found calumenin mRNA to decline in response to heat stress73. Calbindins (Cbp53, −2.5 FC) are a family of proteins that sense Ca2+ concentration and serve as buffers by sequestering intracellular Ca2+74. Given the upregulation of multiple ER stress mechanisms and the role of Ca2+ as a direct messenger between the ER and mitochondria, our data are consistent with a role for Ca2+ homeostasis in the control of cnidarian thermal stress. Oxidative stress and apoptosis Redox and protein homeostasis mechanisms overlap and interact with one another, and oxidative stress stimulates a major signaling pathway that may integrate with other stress signals,


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such as Hsps, through redox control5,17,75. Common cellular reactions involved in regulating oxidative stress involve superoxide dismutases, catalases, glutathione S-transferases (GSTs), and thioredoxins. The levels of two GSTs increased in heat-shocked Aiptasia (1.5 and 2.4 FC), as did two thioredoxin-domain containing PDIs, PDIA4 and ERP44 (2.7 and 1.0 FC, respectively; Table 4). Notably, ROS are generated as a result of PDI protein folding activity (Fig. 3C), which catalyzes the formation and disruption of cysteine-cysteine disulfide bonds in the ER76. Additionally, several Hsps, particularly Hsp60, have been demonstrated to mediate cell stress signaling, activate the innate immune response, and induce NO production77. Although caspase activity has been demonstrated to increase in response to thermal shock in Aiptasia13, and multiple caspases were identified in the Aiptasia proteome here, none exhibited altered abundance between treatments. This may reflect the nature of caspase regulation; caspases are constitutively synthesized and only activated when necessary to alleviate ROS stress3. Superoxide dismutase was also detected, but showed no differential abundance. Mitochondria are a major site of ROS generation as a consequence of their oxidative phosphorylation activity3 (Fig. 3G), and our data suggest changes in response to heat shock that may alter ROS generation. NDUFS1 is the largest subunit of complex I, the first complex of the mitochondrial oxidative phosphorylation electron transport chain78, and NDUFS1 abundance was 1.5 FC greater in heat-shocked anemones (Table 4). Furthermore, complex I can also transfer electrons from NADH/FADH to ubiquinone, and the ubiquinone redox state of the host tissue (not the algal cells) of the coral Acropora millepora has been demonstrated to be highly responsive to hyperthermal stress79. Greater NDUFS1 abundance following heat shock may support an increased rate of respiratory electron flow, and therefore the production of ROS (Fig. 3G). The NDUFS1 complex is involved in apoptosis due to its cleavage by “executioner”



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caspases, such as caspase-3, which disrupt mitochondrial electron flow and promote ROS generation, triggering apoptosis80. Furthermore, mitochondrial 3-mercaptopyruvate sulfurtransferase (3-MST) abundance increased in heat-shocked Aiptasia (1.0 FC). 3-MST produces hydrogen sulfide, which in low concentrations provides electrons that support mitochondrial electron flow coupled to aerobic respiration81. It also enhances glutathione (GSH) production and increases mitochondrial resilience to oxidative stress82. The increased abundances of NDUFS1 and 3-MST indicate potential increases in both ROS generation and ATP production to protect against ROS toxicity. Metabolic responses to thermal shock Maintaining homeostasis in response to thermal change requires shifts in metabolism to accommodate changes in the organism’s energy requirements (e.g. ATP, NADH) or of the flux of different metabolite classes (e.g. amino acids, lipids), and our study reveals changes in multiple metabolic pathways in response to thermal shock. Enzymes that catalyze rate-limiting reactions in central metabolism are likely to be tightly regulated, and an increase in tricarboxylic acid (TCA) cycle activity is a highly conserved response to cellular stress5. Three enzymes of the TCA cycle were found to increase in heat shocked Aiptasia: citrate synthase (2.9 FC), isocitrate dehydrogenase (IDH, 1.2 FC), and isocitrate lyase (0.9 FC). Citrate synthase is thermally sensitive and stabilized by Hsp9083, while IDH, also a member of the conserved MSP, catalyzes the rate-limiting step of the TCA cycle and is one of the few sources of NADPH in animal cells5. Isocitrate lyase is part of the glyoxylate cycle, which short-circuits the TCA cycle to enable enhanced utilization of 2-carbon compounds, such as acetyl-CoA from fatty acid catabolism84. Because antioxidant and macromolecule repair mechanisms require significant ATP and


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NADPH, the increased ATP availability from elevated TCA cycle activity and oxidative phosphorylation may support Hsp functions. Increased NADPH production confers resistance to ROS and NO stress by regenerating reduced antioxidants, and the primary source of NADPH in animals is the pentose phosphate pathway (Fig. 3H), two enzymes of which, transaldolase and transketolase, increased in abundance after heat shock (0.7 and 1.0 FC, respectively)85–87. Furthermore, two oxidoreductases that catalyze NADPH regeneration, L-xylulose reductase and hydroxysteroid dehydrogenase-like protein 2, were elevated (2.3 and 1.0 FC, respectively)88,89. Much of the NADPH produced by these mechanisms is used to regenerate the antioxidant GSH86 (Fig. 3H) and several enzymes of the GSH-producing folate and methionine cycles increased in abundance in response to heat shock. GSH is synthesized via serine90, which is generated from glycolysis by phosphoglycerate dehydrogenase (PHGDH, 2.8 FC) and serine hydroxymethyltransferase (SHMT2, 2.0 FC). SHMT2 and PHGDH are also essential for maintaining mitochondrial redox homeostasis through the production of NADPH91. Methionine adenyltransferase (MAT, 2.7 FC) catalyzes the rate-limiting step of the methionine cycle, producing s-adenosylmethionine (SAMe), the primary methyl donor in eukaryotic cells and an intermediate in GSH biosynthesis. In contrast, betaine hydroxymethyltransferase (−4.6 FC) synthesizes the alternative product of the methionine cycle and was reduced following heat shock. SAMe concentrations are maintained by adenosine kinase92, which was also increased following heat shock (1.7 FC). Phosphoethanolamine nmethyltransferase uses SAMe as a substrate and plays a role in glycerophospholipid synthesis90, and its increased abundance (3.6 FC) may reflect the need to regenerate host membranes damaged by ROS. Elevation of these enzymes suggests increases in NADPH supply for antioxidant mechanisms following heat shock.



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Proteome reorganization of heat-shocked Aiptasia Changes in the protein abundances of heat-shocked Aiptasia signify the induction of multiple stress-response pathways, particularly those involved in protein repair, degradation and re-synthesis. The concerted upregulation of multiple chaperones and protein folding/re-folding mechanisms demonstrates that thermal stress is felt acutely at the ER. The marked reduction in the levels of many otherwise highly abundant cytoskeletal proteins after 24 h of heat shock, combined with the simultaneous increase in several antioxidant mechanisms (e.g. GSTs, PDIs) and enzymes that supply them with reductant (ATP and NADPH) indicates that generalized oxidative damage occurs in response to heat shock. Whether or not these ROS originated from the algal symbiont or were endogenously produced cannot be definitively determined, but endogenous ROS stress would most likely be generated from mitochondrial activity and the observed changes in Ca2+ homeostasis proteins support this view. Excess oxidative damage to mitochondria induces apoptosis, and while this may have occurred in a subset of host cells, mitochondrial proteins were either unchanged or upregulated overall, eliminating the possibility of wholesale mitochondrial degradation. We propose that thermal shock resulted in elevated ROS production by the host, causing widespread protein damage, which was followed by the induction of CSRs to repair, degrade or re-fold damaged proteins (Fig. 3), an interpretation supported by GO term analysis (Fig. 2). The magnitude of the insult, likely taxing to the ER, required increased levels of multiple chaperones to facilitate correct protein folding as well as the induction of ERAD and the UPR to degrade misfolded proteins. The large decline in the abundances of actin and other structural proteins over a relatively short time-scale (24 h), combined with the lack of an obvious biological function for such a


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response, is indicative of elevated protein degradation, impaired protein synthesis, or a combination of the two. We propose that the dramatic loss of cytoskeletal proteins is a result of widespread inhibition of protein synthesis and folding due to ER stress, enhanced by accelerated protein degradation. The ~50% reduction in cytoskeletal proteins after 24 h of heat shock is in line with a previously reported proteolysis capacity of the 26S proteasome of 3-4% degradation of total cell protein per hour56. This experiment was not designed to assess protein turnover per se, and therefore we cannot be definitive in our interpretation of the mechanism of protein degradation. Reductions in epidermal and gastrodermal layer thickness of approximately 25% have previously been described in the coral Acropora aspera after exposure to temperature increases of a smaller magnitude than those imposed here19. This is in accord with our observation that heat-shocked anemones exhibited diminution of four protein toxins (Table 4), presumably localized to the cnidocytes of the epidermal layer, which do not harbor algal symbionts and therefore would not be directly exposed to algal-derived ROS. Hanes and Kempf illustrated the appearance of autophagic structures in Aiptasia tissue after 48 h of heat shock, indicating that in addition to ERAD and proteasome activity, autophagy may play a role in restructuring the host cytoskeleton if the thermal stress is sustained30. Furthermore, Sawyer and Muscatine demonstrated host cell detachment during thermal and Ca2+-disruption stress70. Detachment of whole cells may play a role in the bleaching response at the organismal level93, but, as it would have a universal effect on all cell proteins, it seems unlikely to explain the cytoskeletal modifications detailed here. The CSRs and corresponding changes in protein abundance described here are congruent with previous studies in symbiotic cnidarians. Larvae of Acropora millepora exhibited upregulation of Hsp90 under short-term (4 h) thermal stress but not under long-term stress94.



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Additionally, an analysis of the A. millepora transcriptome revealed greater changes in the gene expression of heat-shocked corals compared with those that had been allowed to thermally acclimate, but the transcript profiles of the thermally-shocked corals showed little overlap with our dataset73. Proteomic analysis of thermally stressed A. microphthalma identified 11 upregulated proteins, including multiple Ca2+-sensing proteins95. Furthermore, DeSalvo et al. detected changes in transcripts encoding Ca2+-binding proteins in bleached corals69, suggesting that disruption of Ca2+ homeostasis, potentially resulting from ROS or NO production, induces organelle damage and cytoskeletal rearrangement. Our data show that these and other CSR mechanisms are not simply a result of elevated temperature but rather are indicative of a rapid disruption of cellular homeostasis prior to algal photoinhibition.

Conclusions The independent thermal susceptibility of the cnidarian host, the dinoflagellate symbiont and different host/symbiont symbioses is an area of active research. ROS generation is considered a likely factor in the bleaching response, and the fact that symbiont abundance and photosynthetic efficiency remained unchanged in our experiment, even during short-term thermal shock, suggests that ROS or NOS were host-derived and were generated before algal stress or bleaching was detected. Aiptasia exhibited considerable capacity to maintain homeostasis with little change in protein abundances under gradually elevated, sustained high temperatures. Short-term heat shock, however, disrupted proteostasis and induced multiple mechanisms to promote protein folding and to degrade improperly folded proteins, indicating an intense impact on ER functions. Based on the observations of reduced cytoskeletal protein abundance and elevated ER protein folding mechanisms, the host appeared to be in the midst of


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large-scale protein reorganization in order to repair damage incurred by the heat shock. The initial mechanism of thermal disruption cannot be identified from this study, but oxidative stress caused by increased production of ROS or NOS seems likely. The cnidarian ER is both a generator and mediator of ROS, and likely plays a central role in maintaining host homeostasis and symbiosis stability during thermal stress..

Author Contributions The project was conceived by CO and SD and conducted with the assistance of ED and LP. Technical assistance and expertise were provided by LP. Statistical analysis was provided by SW. Analysis and interpretation were contributed by CO, VW, AG, and SD. The manuscript was written by CO with insight, editing, and review from ED, LP, SW, VW, AG, and SD. Funding was acquired by SD, VW, and AG.

Acknowledgements This project was funded by the Marsden Fund of the Royal Society of New Zealand, grant number 1202, to SD, VW, and AG. The authors declare no conflicts of interest with the publication of this research.

Supporting Information Figure S1. Grand sums of all total ion current values for all identified proteins per technical replicate. Table S1. Database accession and total ion current value for each identified protein cluster.



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Table S2.Protein cluster report including identification probabilities, sequence coverage, and numbers of unique peptides and spectra. Table S3. Protein clusters that were significantly differentially abundant between treatments, including BLAST statistics, UniprotKB entries, and log2 fold change values.

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Table 1. The 25 most abundant proteins in Aiptasia. % TIC, Acclimated Treatments

% TIC, Heat Shock Treatment

28.47 2.85 1.81

15.74 2.83 1.73

1.62 1.63 1.55

1.46 1.33 1.38

1.41 1.17 1.02 0.70 0.58

0.81 1.06 0.80 0.88 0.75

0.57 0.58 0.46 0.55 0.48 0.47 0.45

0.62 0.60 0.83 0.45 0.62 0.50 0.52

0.45

0.25

0.40 0.37 0.30

0.31 0.35 0.36

0.36 0.29 0.23

0.18 0.24 0.43

Annotation

UniProtKB Accession

E-value

Beta actin*

A0A0A1G3Q1

Tubulin beta chain Myosin heavy chain, striated muscle

P11833 P24733

Golgin subfamily B member 1

Q14789

Failed axon connections homolog Tubulin alpha-1 chain* Zona pellucida domain-containing protein* Spectrin alpha chain, non-erythrocytic 1* Filamin-A* Protein disulfide-isomerase Glutamate dehydrogenase

D3ZAT9 Q8T6A5

1.4E−10 7 0 0 1.5E−10 2 8.5E−40 0

G8HTB6

6.5E−83

P07751 Q8BTM8 P07237 A7SE06

14-3-3 protein epsilon

P62262

Calpain-B Heat shock cognate 71 kDa protein Tropomyosin-1, isoforms 9A/A/B* Ectin Spectrin beta chain, non-erythrocytic 1 Hemicentin-1

Q9VT65 P19120 Q95VA8 B3EWZ8 Q01082 Q96RW7-2

Mucin-2*

Q62635

Tyrosine decarboxylase, putative*

A0A0G3CHC0

Arginine kinase Clathrin heavy chain 1

O15992 P49951

Golgin subfamily B member 1 isoform 4

Q14789-4

Tight junction protein ZO-1* Myosin-10

Q07157-2 Q27991

0 0 0 0 4.5E−10 2 0 0 2.8E−64 2.4E−06 0 9.2E−35 1.6E−16 8 1.4E−14 4 0 0 1.9E−13 9 2.4E−80 0

*Proteins that were differentially abundant between (25 °C) and heat shock (33.5 °C) treatments. Shaded proteins are cytoskeletal, structural, or muscle proteins. “Acclimated treatments” include anemones acclimated to 25 °C, 30 °C, and 33.5 °C, while “heat shock” indicates anemones transferred from 25 °C to 33.5 °C for 24 h. TIC = Total ion current.


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Table 2. The amount of differentially abundant (q < 0.1) proteins between the thermal treatments based on false discovery rates. Treatment

25 °C

25 °C 8 30 °C 9 33.5 °C 104 33.5 °C HS HS = heat shock, 24 h.

30 °C

33.5 °C

3 49

74

33.5 °C HS

-



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Table 3. Differentially abundant proteins between Aiptasia acclimated to control (25 °C) and high temperature (30 °C and 33.5 °C) conditions for two weeks. Fold Annotation Changea 25 °C versus 30 °C (Acclimated) 1.80 Isochorismatase domain-containing protein 1 1.66 Predicted protein 1.04 Fructosamine 3 kinase −1.06 Inactive pancreatic lipase-related protein 1 −1.13 Vasodilator-stimulated phosphoprotein (VASP) −1.41 DsRNase 4 −1.95 Putative uncharacterized protein 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase −2.41 gamma-1 25 °C versus 33.5 °C (Acclimated) 1.79 Isochorismatase domain-containing protein 2 NADH-ubiquinone oxidoreductase 75 kDa subunit 1.48 (NDUFS1) 1.10 Beta gamma crystallin isoform 8 Pyruvate carboxylase −1.07 Endoplasmic reticulum resident protein 44 −1.09 Phosphoinositide phospholipase C eta 2 −1.09 Elongation factor 2 −1.33 Acetylcholinesterase −1.47 Elongation factor 1 gamma −1.55 a

UniProtKB Accession

E-value

Q5PQ71 A7SA07 Q8K274 P54316 P70460 X2JC21 E9G363

3.7E−72 3.7E−40 1.9E−94 1.7E−62 1.5E−69 1.8E−17 4.3E−17

Q62077

0

Q5PQ71

3.6E−72

P15690 A8C9L8 Q29RK2 Q9D1Q6 A2AP18-5 Q3SYU2 Q92035 Q6PE25

0 2.9E−28 0 1.3E−96 4.9E−69 0 1.8E−38 1.6E−56

“Fold change” represents the log2 ratio of proteins from anemones maintained at 25 °C versus

those from anemones subjected to elevated temperatures.


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Table 4. Selected differentially abundant (q < 0.1) proteins between Aiptasia acclimated to control (25 °C) and heat-shocked (33.5 °C, 24 h) conditions. Fold Annotation Changea Cytoskeleton, structure and muscleb Tubulin alpha-1A −0.56 Spectrin alpha chain, non-erythrocytic 1 −0.58 Talin-2 −0.67 Vitelline membrane outer layer protein 1 homolog −0.69 Tropomyosin-1, isoforms 9A/A/B −0.69 Actin-related protein 2 −0.69 Talin-2 −0.71 Myosin light chain 6B −0.74 EGF and laminin G domain-containing protein −0.76 Filamin-A −0.76 Myosin-2 heavy chain −0.92 Zona pellucida domain-containing protein −1.12 Beta actin −1.12 Mucin-2 −1.25 Talin-1 −1.47 Myosin light chain kinase −1.56 Advillin −2.06 Myosin, light chain regulatory protein −2.74 Vinexin −3.06 Heat stress and protein folding 4.47 Calumenin-B (CALU) 2.77 Stress-70 protein, mitochondrial (HspA9, Hsp70) 2.76 Beta coatomer (COPI) 2.68 Protein disulfide-isomerase A4 (PDIA4) 2.49 T-complex protein 1 subunit beta (CCT2) 1.79 Prohibitin-2 (PHB2) 1.33 T-complex protein 1 subunit beta (CCT2) 1.22 Binding immunoglobulin protein (BiP, Hsp70) Dolichyl-diphosphooligosaccharide--protein 1.17 glycosyltransferase subunit 1 (OST1) 1.17 T-complex protein 1 subunit alpha (CCT1) 1.12 Endoplasmin (Hsp90B1) 1.10 T-complex protein 1 subunit epsilon (CCT5) 1.07 Transitional endoplasmic reticulum ATPase (VCP) 1.06 Heat shock protein Hsp90-alpha (Hsp90A) 1.03 Endoplasmic reticulum resident protein 44 (Erp44) 0.61 60kDa heat shock protein, mitochondrial (Hsp60) Oxidative stress


UniProtKB Accession

E-Value

Q8T6A5 P07751 Q9Y4G6 Q5SXG7 Q95VA8 Q5M7U6 Q9Y4G6 P14649 B8UU78 Q8BTM8 P08799 G8HTB6 A0A0A1G3Q1 Q62635 P54939 Q6PDN3-2 O88398 P40423 O60504

0 0 0 2.4E−24 2.8E−64 0 0 8.7E−26 0 0 7.5E−09 6.5E−83 1.4E−107 1.6E−168 3.5E−180 0 1.1E−114 5.4E−28 1.4E−60

Q7SXV9 Q5R511 P23514 P13667 Q3ZBH0

3.1E−55 0 0 0 0

P80314 Q90593

1.1E−143 0

Q91YQ5

0

Q9W790 P08110 Q4R6V2 P46462 P07900 Q9D1Q6 O02649

0 0 0 0 0 1.3E−96 0


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2.77 Stress-70 protein, mitochondrial (HspA9, Hsp70) 2.68 Protein disulfide-isomerase A4 (PDI) 2.36 Glutathione S-transferase U25 1.83 NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1) 1.49 Glutathione S-transferase 1 (GSTσ) 1.03 Endoplasmic reticulum resident protein 44 0.99 Hydroxysteroid dehydrogenase-like protein 2 (HSDL-2) −0.74 Indole-3-acetaldehyde oxidase Core metabolism 2.90 Citrate synthase, mitochondrial 2.75 D-3-phosphoglycerate dehydrogenase (PHGDH) 2.30 L-xylulose reductase (DCXR) 2.16 Fructose-1,6-bisphosphatase 1 1.83 NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1) 1.17 Isocitrate dehydrogenase [NADP], mitochondrial (IDH) 1.03 Transketolase-like protein 2 0.93 Isocitrate lyase (ICL) 0.65 Transaldolase −1.18 Glutamine synthetase (GS) Lipid transport, metabolism, and beta oxidation 3.59 Phosphoethanolamine N-methyltransferase 3 0.90 Trifunctional enzyme subunit β (ACAA2) Enoyl-CoA hydratase, mitochondrial (SCEH) −1.29 Inactive pancreatic lipase-related protein 1 −1.60 Inactive pancreatic lipase-related protein 1 −2.00 One-carbon metabolism and s-adenosylmethionine biosynthesis 2.71 S-adenosylmethionine synthase (MAT) 2.00 Serine hydroxymethyltransferase (SHMT2) 1.70 Adenosine kinase 2 (ADK2) 0.95 3-mercaptopyruvate sulfurtransferase isoform 2 (3-MST) −4.64 Betaine-homocysteine S-methyltransferase (BHMT) Calcium binding 4.47 Calumenin-B (CALU) 1.03 Transketolase-like protein 2 −0.14 Beta gamma crystallin isoform 8 −0.74 Myosin light chain 6B −0.76 EGF and laminin G domain-containing protein −1.56 Myosin light chain kinase −2.06 Advillin −2.32 Hemolysin-type calcium-binding protein −2.47 Calbindin-32 (Cbp53E) −2.74 Myosin regulatory light chain sqh Toxins −1.51 Delta-aiptatoxin-Adi1a


Q5R511 P13667 Q9SHH7 P15690 P46436 Q9D1Q6 Q6P5L8 Q7G192

0 0 6.6E−27 0 1.0E−37 1.3E−96 0 0

Q16P20 Q5R7M2 Q7Z4W1 Q3SZB7 P15690 Q4R502 Q9D4D4 Q9K9H0 Q9EQS0 Q6S3M2

0 2.0E−114 2.7E−51 2.7E−133 0 0 0 0 1.3E−152 0

Q944H0 P13437 P14604 P54316 Q5BKQ4

3.4E−96 1.4E−179 6.5E−113 1.7E−62 5.6E−64

A7SEP6 P34897 Q9LZG0 Q99J99 Q5XGM3

0 0 2.4E−134 7.7E−79 1.4E−175

Q7SXV9 Q9D4D4 A8C9L8 P14649 B8UU78 Q6PDN3-2 O88398 B9NTX7 P41044 P40423

3.1E−55 0 2.9E−28 8.7E−26 0 0 1.1E−114 1.1E−40 1.4E−31 5.4E−28

E3P6S4

1.5E−26

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−1.94 −2.18 −2.32 a

Delta-alicitoxin-Pse2b Acetylcholinesterase Hemolysin-type calcium-binding protein

P58912 Q92035 B9NTX7

0 1.8E−38 1.1E−40

“Fold change” represents the log2 ratio of proteins from heat shocked anemones to those of

control anemones. bFunctional categories are not exclusive; proteins may appear in multiple categories. Red and blue indicate increased and decreased abundance in heat shocked anemones, respectively.



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Figure Legends Figure 1. Density and quantum yield of photosystem II chlorophyll fluorescence of algal symbionts in Aiptasia. Anemones were kept at 25 °C (25 °C), acclimated to 30 °C (30 °C), acclimated to 33.5 °C (33.5 °C), or heat-shocked at 33.5 °C for 24 h without thermal acclimation (33.5 °C HS). A) Algal cell density at the time of proteome sampling, n = 6. B) Dark-adapted quantum yield of photosystem II chlorophyll fluorescence of Aiptasia symbionts prior to and during the thermal treatment, n = 3–6 per time point. Day 0 (dashed line) indicates the end of the acclimation period and the beginning of the thermal treatments. Figure 2. Counts of proteins that were differentially abundant (q < 0.1) between control (25 °C) and heat-shocked (33.5 °C, 24 h) Aiptasia, grouped by biological process gene ontology terms. A) More abundant proteins in heat-shocked Aiptasia (minimum four shown) or B) less abundant proteins in heat-shocked anemones (minimum three shown). Figure 3. Cellular processes proposed to be induced by thermal shock in Aiptasia. A) Cytoskeletal proteins are damaged by reactive oxygen species (ROS) exposure (red regions). The damaged proteins are ubiquinated (green circles) and targeted to the proteasome for degradation. The folding of new cytoskeleton proteins is stabilized by chaperonins (CCT). B) Nascent proteins either fold correctly (blue) or are misfolded (red). Misfolding rates increase under high temperatures, resulting in an accumulation of misfolded proteins and the induction of the unfolded protein response. C) Misfolded proteins are identified by heat shock proteins (Hsps), such as Hsp90, and are either repaired or unfolded for retrotranslocation from the endoplasmic reticulum (ER), which is then followed by ubiquination and subsequent degradation by the proteasome (ER-associated degradation, ERAD). Protein unfolding by protein disulfide isomerase results in ROS generation. D) Increases in coatomer (COPI) protein reflect an


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increased amount of COPI vesicle cycling between the ER and Golgi apparatus, which increases the influx of damaged or misfolded proteins into the ER. E) Proteins that are unable to be refolded are exported from the ER, ubiquinated and transported to the proteasome for degradation via ERAD. F) The secondary messenger Ca2+ is exchanged between the ER and mitochondria through tight junctions stabilized by HspA9. Transfer of Ca2+ from the ER to the mitochondria increases during ER stress, stimulating mitochondrial respiration and potentially inducing apoptosis. G) Upregulation of both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation increases the reductant available for antioxidant mechanisms as well as the generation by NADH-ubiquinone oxidoreductase (NDUFS1) of ROS, some of which may leak from the mitochondria. H) The antioxidant glutathione (GSH) repairs oxidized proteins, and its oxidized form (GSSG) is reduced by NADPH generated by the pentose phosphate pathway (PPP). GSH is synthesized as a result of the methionine/S-adenosyl methionine (SAMe) cycle, enzymes of which are upregulated by increased temperatures.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Density and quantum yield of photosystem II chlorophyll fluorescence of algal symbionts in Aiptasia. Anemones were kept at 25 °C (25 °C), acclimated to 30 °C (30 °C), acclimated to 33.5 °C (33.5 °C), or heat-shocked at 33.5 °C for 24 h without thermal acclimation (33.5 °C HS). A) Algal cell density at the time of proteome sampling, n = 6. B) Dark-adapted quantum yield of photosystem II chlorophyll fluorescence of Aiptasia symbionts prior to and during the thermal treatment, n = 3–6 per time point. Day 0 (dashed line) indicates the end of the acclimation period and the beginning of the thermal treatments. 132x227mm (300 x 300 DPI)


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Figure 2. Counts of proteins that were differentially abundant (q < 0.1) between control (25 °C) and heatshocked (33.5 °C, 24 h) Aiptasia, grouped by biological process gene ontology terms. A) More abundant proteins in heat-shocked Aiptasia (minimum four shown) or B) less abundant proteins in heat-shocked anemones (minimum three shown). 257x244mm (300 x 300 DPI)



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Figure 3. Cellular processes proposed to be induced by thermal shock in Aiptasia. A) Cytoskeletal proteins are damaged by reactive oxygen species (ROS) exposure (red regions). The damaged proteins are ubiquinated (green circles) and targeted to the proteasome for degradation. The folding of new cytoskeleton proteins is stabilized by chaperonins (CCT). B) Nascent proteins either fold correctly (blue) or are misfolded (red). Misfolding rates increase under high temperatures, resulting in an accumulation of misfolded proteins and the induction of the unfolded protein response. C) Misfolded proteins are identified by heat shock proteins (Hsps), such as Hsp90, and are either repaired or unfolded for retrotranslocation from the endoplasmic reticulum (ER), which is then followed by ubiquination and subsequent degradation by the proteasome (ER-associated degradation, ERAD). Protein unfolding by protein disulfide isomerase results in ROS generation. D) Increases in coatomer (COPI) protein reflect an increased amount of COPI vesicle cycling between the ER and Golgi apparatus, which increases the influx of damaged or misfolded proteins into the ER. E) Proteins that are unable to be re-folded are exported from the ER, ubiquinated and transported to the proteasome for degradation via ERAD. F) The secondary messenger Ca2+ is exchanged between the ER and mitochondria through tight junctions stabilized by HspA9. Transfer of Ca2+ from the ER to the mitochondria increases during ER stress, stimulating mitochondrial respiration and potentially inducing apoptosis. G) Upregulation of both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation increases the reductant available for antioxidant mechanisms as well as the generation by NADH-ubiquinone oxidoreductase (NDUFS1) of ROS, some of which may leak from the mitochondria. H) The antioxidant glutathione (GSH) repairs oxidized proteins, and its oxidized form (GSSG) is reduced by NADPH generated by the pentose phosphate pathway (PPP). GSH is synthesized as a result of the methionine/S-adenosyl methionine (SAMe) cycle, enzymes of which are upregulated by increased temperatures. 972x737mm (96 x 96 DPI)


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For TOC only. 1344x746mm (96 x 96 DPI)


Thermal Shock Induces Host Proteostasis Disruption and

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