Abstract
Cells have elaborated a complex strategy to maintain protein homeostasis under physiological as well as stress conditions with the aim to ensure the smooth functioning of vital processes and producing healthy offspring. Impairment of one of the most important processes in living cells, translation, might have serious consequences including various brain disorders in humans. Here, we describe a variant of the translation initiation factor eIF3a, Rpg1-3, mutated in its PCI domain that displays an attenuated translation efficiency and formation of reversible assemblies at physiological growth conditions. Rpg1-3–GFP assemblies are not sequestered within mother cells only as usual for misfolded-protein aggregates and are freely transmitted from the mother cell into the bud although they are of non-amyloid nature. Their bud-directed transmission and the active movement within the cell area depend on the intact actin cytoskeleton and the related molecular motor Myo2. Mutations in the Rpg1-3 protein render not only eIF3a but, more importantly, also the eIF3 core complex prone to aggregation that is potentiated by the limited availability of Hsp70 and Hsp40 chaperones. Our results open the way to understand mechanisms yeast cells employ to cope with malfunction and aggregation of essential proteins and their complexes.
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Acknowledgements
We are grateful to Prof. Robert Singer, Dr. Mark Ashe, Prof. Gary Jones and Prof. Yves Barral for sharing strains and plasmids with us. We also thank Jana Vojtova for critical comments on the manuscript, Lenka Novakova for her technical assistance, and other members of the Hasek lab for helpful discussions.
Funding
This research was supported by the grant from the Czech Science Foundation CSF16-05497S (J.H.) and the Grant Agency of the Charles University GACU1180213 (L.S.)
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IM, JH, and LS conceived and designed the experiments. LS and IM performed the experiments, analyzed data and prepared the manuscript. JH conceived the experiments, contributed to the preparation of the manuscript and approved its final version.
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294_2019_940_MOESM1_ESM.eps
Supplementary material 1: Fig. S1. Polysome profile analysis of rpg1-3 (CRY1679) and wild-type RPG1 (CRY1683) cells exponentially growing in YPD a at 25°C or b after the shift to 37°C for 4 h. Positions of the 40S, 60S, monosome, and polysome are indicated by arrows. The number representing the polysome to monosome area ratio (P/M) is given above the profiles. c The shift to the restrictive temperature does not cause aggregation either Rpg1–GFP or Rpg1-3–GFP. Exponentially growing cells expressing wild-type Rpg1–GFP (CRY1683) or mutant Rpg1-3–GFP (CRY1679) were transferred from 25 °C to 37 °C for 1 h. Scale bars – 4 μm (EPS 3979 KB)
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Supplementary material 2: Fig. S2. The movement of dynamic Rpg1-3–GFP foci does not depend on microtubules. a In exponentially growing cells co-expressing Rpg1-3–GFP and mCherry-Tub1 (CRY1990), α-tubulin decorated spindle and cytoplasmic microtubules while in cells treated with nocodazole α-tubulin accumulated at spindle pole bodies (SPBs) only. b Time-lapse series of cells with Rpg1-3–GFP foci treated with nocodazole were recorded in 4.5s-interval then trajectories were created and alpha coefficient, velocity, and MSD were calculated. The white arrow marks the analyzed Rpg1-3–GFP assembly and the circle indicates its final position. The dashed lines highlight the cell border. M - mother cell, D - daughter cell. Values of the alpha coefficient, velocity and MSD describe the presented trajectory. The dashed line in the plot represents a pure random walk and the solid line the result for the displayed trajectory. Scale bars – 4 μm (EPS 3801 KB)
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Supplementary material 3: Fig. S3. Latrunculin B depolymerizes actin filaments and impairs the directionality of trajectories of Rpg1-3–GFP dynamic foci. a Actin filaments were visible in exponentially growing cells expressing Rpg1-3–GFP and the actin-filament marker protein Abp140–yTagRFP-T (CRY2077), whereas they were absent in cells treated with latrunculin B and Abp140–yTagRFP-T accumulated at actin patches. Scale bars – 4 μm. b Trajectories of Rpg1-3–GFP foci under physiological conditions (25 °C) within the mother cells clearly monitored the directionality of the Rpg1-3–GFP foci movement. Treatment with latrunculin B abolished the directionality of trajectories of Rpg1-3–GFP foci within the mother cells (EPS 3991 KB)
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Supplementary material 4: Fig. S4. Myo2 but not Myo4 and She3 is implicated in the directional movement of dynamic Rpg1-3–GFP foci. a Particle-tracking analysis of time-lapse series (recorded in 2-s intervals) of the double deletion myo4∆, she3∆ strain (CRY2566). White arrows mark the analyzed Rpg1-3–GFP assembly and the colored circle indicates its final position. The dashed lines highlight the cell border. M - mother cell, D - daughter cell, BDM - bud-directed movement. Values of the alpha coefficient, velocity and MSD describe the presented trajectory. The dashed line in the plot represents a pure random walk and the solid line the result for the displayed trajectory. b Heat inactivation of the myo2-66 mutant (CRY2782) (37 °C, 1 h) changed the directionality of Rpg1-3–GFP foci trajectories within mother cells. c In contrast, cultivation of rpg1-3 (CRY1679) cells carrying the wild-type MYO2 at 37 °C for 1 h did not have any impact on the BDM or on the oriented movement of Rpg1-3–GFP foci within mother cells. Time-lapse series were taken in 2-s intervals and foci trajectories were created. The white arrow marks the analyzed Rpg1-3–GFP assembly and the colored circle indicates its final position. The dashed lines highlight the cell border. M - mother cell, D - daughter cell. Values of the alpha coefficient, velocity and MSD describe the presented trajectory. The dashed line in all plots represents a pure random walk and the solid line the result for the displayed trajectory. Scale bars – 4 μm. d Temperature inactivation of myo2-66 did not lead to an increased formation of Rpg1-3–GFP assemblies. The number of cells with foci was determined in cell cultures (CRY2782) exponentially growing in YPD at 25°C or after their shift to 37 °C for 1 h. Error bars represent SD calculated from three independent experiments with more than 1 000 cells analyzed in each (EPS 4757 KB)
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Supplementary material 5: Fig. S5. Assemblies of Rpg1-3–GFP associate with ER and mitochondria. To demonstrate better the association of Rpg1-3–GFP foci with ER and mitochondria in the mother cell and in the bud we show consecutive optical sections (layers) from a Z-stack. a Distribution of Rpg1-3–GFP foci in the strain expressing Rpg1-3–GFP and the ER-marker HDEL-DsRed (CRY2336). Association of Rpg1-3–GFP foci with ER in the bud (layers 6-8) and in the mother cell (layers 13-15). b Association of Rpg1-3-GFP foci with mitochondria and ER in triple fluorescently labeled cells (mitochondria – pYX142-mito-mTagBFP, ER – HDEL-DsRed, Rpg1-3–GFP) (CRY1936). Scale bars – 4 μm (EPS 7704 KB)
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Supplementary material 6: Fig. S6. Immobile Rpg1-3–GFP foci are decorated by Hsp104 in post-diauxic cells. a Cells co-expressing Rpg1-3–GFP and Hsp104–yTagRFP-T (CRY1861) were grown for more than 15 h in YPD at 25 °C. b Time-lapse series were recorded in 4.1-s intervals and trajectories were created. White bold and dashed arrows mark the analyzed Rpg1-3–GFP foci. The alpha coefficient, velocity, and MSD were calculated. The dashed line in the plot represents a pure random walk and the solid line the result for the displayed trajectory. c Proteasome inhibition by MG132 does not cause sequestration of Rpg1-3–GFP foci. Exponentially growing cells co-expressing Rpg1-3–GFP and Hsp104–yTagRFP-T and the pdr5 deletion (CRY2912) were treated with 80 μM MG132 for 1 h before observation. Scale bars – 4 μm (EPS 6123 KB)
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Supplementary material 7: Fig. S7. Members of the eIF3 core complex are present in Rpg1-3–GFP assemblies even at the restrictive temperature. a Exponentially growing cells were cultivated in YPD at 25 °C and then shifted to 37 °C for 1 h. Colocalization of Rpg1-3–GFP with members of the eIF3 core complex—Nip1–mRFP (CRY1888), Prt1–yTagRFP-T (CRY1874), and Hcr1–yTagRFP-T (CRY1873). b Rpg1-3–GFP assemblies are not stress granules. Exponentially growing cells co-expressing Rpg1-3–GFP with Pab1–yTagRFP-T (CRY2439) and Yef3–yTagRFP-T (CRY2018) were analyzed. c Wild-type Rpg1–GFP stays equally distributed in exponentially growing cells co-expressing Rpg1–GFP and Dcp2–yTagRFP-T (CRY1869) or Rpg1–GFP and Xrn1–yTagRFP-T (CRY2297) at 25 °C. Scale bars – 4 μm (EPS 9019 KB)
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Supplementary material 8: Fig. S8. In silico model of a part of the PCI domain of Rpg1 and Rpg1-3 proteins. Structure prediction of a crystallized part ADDIN EN.CITE (Khoshnevis et al. 2014) of the protein molecule encompassing the PCI domain (AA residues 276-494) of the a WT Rpg1 and b the Rpg1-3 mutant proteins was performed by the ITASSER software ADDIN EN.CITE (Roy et al. 2010; Yang et al. 2015; Zhang 2008) and visualized by PyMol (The PyMOL Molecular Graphics System, Version 1.8.x Schröndinger, LLC). Shortened helix 5 due to the deletion of 6 AA residues in the Rpg1-3 mutant ends with D389 (grey) instead with E392 (blue) but D389 (grey) is still able to form hydrogen bonds (yellow) with R363 (cyan) but not with R431 (pink) anymore since the deletion of E392 (dark blue) displaces the helix 7 containing R431 (pink) further away from the helix 5 with D389. The distance between D389 (grey) and R431 (pink) is approximately 3.4-4.5-fold higher than in the case of binding of R431 (pink) with E392 (dark blue). Blue dashed lines depict the distance (EPS 8676 KB)
Supplementary material 9: Online Resource 1a. Rpg1-3–GFP assemblies are transmitted to the progeny. Cells bearing the rpg1-3 mutant (CRY1679) were continuously cultivated in the CellASIC microfluidic system. Images were taken in 90-min intervals. The movie was created in Fiji/ImageJ2 and is played at speed 2 fps. a DIC (AVI 3920 KB)
Supplementary material 10: Online Resource 1b. Rpg1-3–GFP assemblies are transmitted to the progeny. Cells bearing the rpg1-3 mutant (CRY1679) were continuously cultivated in the CellASIC microfluidic system. Images were taken in 90-min intervals. The movie was created in Fiji/ImageJ2 and is played at speed 2 fps. b Rpg1-3–GFP (AVI 3920 KB)
Supplementary material 11: Online Resource 2. Immobile Rpg1-3–GFP assemblies are tethered to ER and mitochondria. Micrographs of triple fluorescently labeled cells (mitochondria – pYX142-mito-mTagBFP, ER – HDEL-DsRed, Rpg1-3–GFP) (CRY1936) were acquired in 9.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at speed 6 fps (AVI 9305 KB)
Supplementary material 12: Online Resource 3a. Rpg1-3–GFP assemblies vibrate near the ER and mitochondria in energy-depleted rpg1-3 cells. Triple fluorescently labeled cells (mitochondria – pYX142-mito-mTagBFP, ER – HDEL-DsRed, Rpg1-3–GFP) (CRY1936) were treated with FCCP (a). Micrographs were acquired in 9.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at speed 6 fps (AVI 8326 KB)
Supplementary material 13: Online Resource 3b. Rpg1-3–GFP assemblies vibrate near the ER and mitochondria in energy-depleted rpg1-3 cells. Triple fluorescently labeled cells (mitochondria – pYX142-mito-mTagBFP, ER – HDEL-DsRed, Rpg1-3–GFP) (CRY1936) were treated with the combination of antimycin A and 2-deoxyglucose (b). Micrographs were acquired in 9.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at speed 6 fps (AVI 9794 KB)
Supplementary material 14: Online Resource 4. Dynamic Rpg1-3–GFP focus follows ER tubules passing through the bud neck. Micrographs of cells expressing Rpg1-3–GFP and the ER-marker HDEL-DsRed (CRY2336) were taken in 4.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at speed 6 fps (AVI 7347 KB)
Supplementary material 15: Online Resource 5a. Members of the eIF3 core complex are moving together with Rpg1-3–GFP in common foci from the mother to the daughter cell. Micrographs of exponentially growing cells co-expressing Rpg1-3–GFP with Nip1–mRFP (CRY1888) (a) were taken in 4.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at 6 fps (AVI 15179 KB)
Supplementary material 16: Online Resource 5b. Members of the eIF3 core complex are moving together with Rpg1-3–GFP in common foci from the mother to the daughter cell. Micrographs of exponentially growing cells co-expressing Rpg1-3–GFP with Hcr1–yTagRFP-T (CRY1873) (b) were taken in 4.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at 6 fps (AVI 4409 KB)
Supplementary material 17: Online Resource 6. Immobile Rpg1-3–GFP foci associate with P-body marker protein Dcp2. Micrographs of exponentially growing cells co-expressing Rpg1-3–GFP with Dcp2–yTagRFP-T (CRY1868) were taken in 4.5-s intervals. The movie was created in Fiji/ImageJ2 and is played at 6fps (AVI 12242 KB)
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Supplementary material 18: Table S1. Doubling time of the wild-type Rpg1-GFP (CRY1683) and the mutant Rpg1-3-GFP (CRY1679) cells. Exponentially growing cells were cultivated in YPD at 25 °C in a microplate reader Reader EON™ Microplate Spectrophotometer (BioTek, USA). Doubling times and S.D. were calculated from three independent experiments (DOC 28 KB)
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Senohrabkova, L., Malcova, I. & Hasek, J. An aggregation-prone mutant of eIF3a forms reversible assemblies escaping spatial control in exponentially growing yeast cells. Curr Genet 65, 919–940 (2019). https://doi.org/10.1007/s00294-019-00940-8
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DOI: https://doi.org/10.1007/s00294-019-00940-8