Faculty Mentor: Adam Hughes (Biochemistry, University of Utah)
Abstract
The dysfunction of the mitochondria is related to a variety of stressors and is a hallmark of aging and age-related disease. As a way of responding to mitochondrial defects, cells use various mitochondrial degradation pathways, one including Mitochondrial-Derived Compartments. MDCs remove specific outer and inner mitochondrial membrane proteins, and require the OMM protein Tom70 for formation. To better understand MDC formation pathways, we investigated the role of ubiquitin by tagging the deubiquitinase (DUB) UL36 to Tom70 in the presence of various metabolic and protein overloading stressors. Through fluorescence microscopy, we found ubiquitin to be significant for the formation of MDCs under cycloheximide and SCM4 overexpression induced stress in yeast cells expressing Tom70-UL36 and MDC marker proteins. We additionally found the DUB to inhibit MDCs rather than only preventing cargo from entering MDCs in yeast cells expressing Tom70- GFP-UL36. These results demonstrate the importance of ubiquitin in MDC formation under specified stressors.
Introduction
Mitochondrial dysfunction is a hallmark of aging and age-related diseases. The mitochondria is integral for cell processes, including, but not limited to, precursor and cofactor biosynthesis, energy production, and intrinsic apoptosis regulation. To maintain mitochondrial homeostasis and keep such functions stable, cells utilize numerous mitochondrial degradation pathways that remove unwanted mitochondria.
Without these pathways, where cells dynamically respond to stress, mitochondrial homeostasis and, in turn, function, can be impaired. For most eukaryotes, this creates vulnerability to age-related conditions, ranging from neurodegenerative disorders, cardiovascular disease, and cancer, among other pathologies linked to aging. Addressing gaps in mitochondrial regulation research is vital for advancing scientific understanding of related conditions. The Hughes Lab identified Mitochondrial- Derived Compartments (MDCs) as a unique cargo-carrying structure that is part of a mitochondrial quality control pathway, which reconfigures the mitochondrial proteome of aged yeast and mammalian cells in response to certain stress. In contrast to other mitochondrial degradation pathways, MDCs are selective for a specific set of outer mitochondrial membrane proteins (OMM) and inner mitochondrial membrane proteins (IMM), which are sequestered for degradation without compromising the entirety of the organelle.
The Hughes Lab additionally found Tom70, an OMM receptor protein and known MDC cargo, to be essential for MDC formation, but the mechanisms by which it contributes to MDC formation remain unclear. Given that ubiquitination is known to be significant in membrane remodeling and protein sorting, we were interested in investigating ubiquitin’s possible involvement in MDC formation by inhibiting ubiquitination of Tom70. The deubiquitinase (DUB) UL36, a protein that removes ubiquitin from target proteins, was tagged to Tom70 in yeast cells. To test the range of MDC inducers that are blocked by Tom70-UL36, MDCs were induced by metabolic or protein- overloading stress in three separate experiments. 1) Amino acid buildup by cycloheximide (CHX) treatment. 2) SRP knockdown by auxin treatment leading to protein overload at the mitochondrial surface. 3) OMM protein overload through SCM4 overexpression (OE) by genetic modification. These experiments used Tim50-mCherry and Tcd2-GFP to visualize MDCs. To investigate whether the DUB blocks MDC formation rather than just preventing cargo from entering MDCs, in a fourth experiment we tagged Tom70-UL36 with GFP and assessed MDCs induced by rapamycin (rap), a standard metabolic inducer.
Results
Experiments 1-3showed that Tcd2-positive MDCs were blocked by the DUB on or near Tom70 when treated with CHX (Fig. 2) or during SCM4 OE (Fig. 3). In yeast cells with SRP knockdown, the inhibition of ubiquitination on or near Tom70 did not block Tcd2 carrying MDCs (Fig. 4). Experiment 4 found the DUB on Tom70 to block Tom70-positive MDCs during rap-induced metabolic stress (Fig. 5). This suggests that the DUB on Tom70 directly inhibits MDC formation, rather than just preventing cargo entry into MDCs. These findings demonstrate ubiquitin’s significance in MDC formation under certain metabolic or protein-overloading stressors, where Tcd2 or Tom70 are cargo. It additionally suggests that ubiquitination is unnecessary for MDC formation caused by SRP knockdown. Future studies should investigate MDC formation pathways during SRP knockdown- induced stress and whether the ubiquitination requirement is specific to Tom70 or if it is the presence of ubiquitin on the mitochondrial surface that is necessary for MDC formation.
Discussion and Future Directions
MDCs are known to form in response to the metabolic stressor rapamycin, which inhibits mTOR signaling pathways and subsequently protein synthesis, and concanamycin A, which inhibits the cell’s ability to correctly store amino acids. Both of these drug treatments lead to a build-up of amino acids in the cells that is followed by MDC formation. When yeast cells expressing Tom70- UL36 were treated with these drugs, MDCs were blocked. Another known MDC inducer, cycloheximide (CHX), similarly causes amino acid build-up by inhibiting protein synthesis in cells; so, it was hypothesized and shown to be true (Fig. 2), that yeast cells containing Tom70-UL36 would block MDCs induced by the metabolic stressor CHX as well. It remains unclear how these metabolic inducers exactly cause MDCs, but it is suggested that amino acid build up caused by these drugs somehow leads to ubiquitination of Tom70 as a way of triggering MDC formation. One possible explanation is that the amino acids somehow block or affect functions of OMM proteins, such as Tom70. Further research could be conducted on the specific pathways that link amino acid buildup to MDC formation as a way to uncover a possible trigger of Tom70’s ubiquitination.
Additionally, amino acid build up has been linked to MDC formation as studied in the Hughes Lab, with research suggesting that a disruption in amino acid homeostasis triggers the remodeling of mitochondria. This would include the removal of Tom70 which is an import receptor for SLC25A, a nutrient carrier that transports amino acids. This mitochondrial remodeling, where nutrient transporters are potentially removed, could be the cell trying to rapidly stop a harmful influx of amino acids or other metabolites from entering the mitochondria.
Overall, our research, in accordance with past studies, indicates ubiquitination to be required for MDC formation caused by stress in the mitochondria, and Tom70 was observed to be ubiquitinated for MDC formation during certain mitochondrial stress.
The inhibition of MDCs in yeast strains expressing Tom70-UL36 is significant in confirming ubiquitination as vital for MDC formation caused by cycloheximide treatment (Fig. 2) and forced SCM4 overexpression (Fig. 3) Results also found MDCs induced by SRP knockdown to not be affected by Tom70-UL36 (Fig. 4), which may suggest MDC formation during SRP knockdown to differ from pathways in experiments that saw MDC blockage. Future research should test for the amount of ubiquitin on the mitochondrial surface in these different stressors, which could be done by isolating the mitochondria from cells in varying conditions, and then using antibodies to analyze ubiquitin levels. Differences may indicate specific ubiquitin amount requirements for specific MDC inducers and may suggest if UL36 was incapable of handling the ubiquitin levels present during SRP knockdown. The translocation of proteins to the ER that is regulated by SRP affects many SRP pathway-dependent proteins and organelles, leading to defects in other cellular processes too, whereas SCM4-OE only creates an overload of that specific protein. If ubiquitin levels were found to be much higher in cells with SRP knockdown, it could be possible that UL36 has a threshold of ubiquitin it can properly remove, rather than the idea that SRP knockdown-induced MDCs do not require ubiquitin.
The final experiment aimed to investigate if the MDC inhibition seen was a genuine blockage of MDC formation or if only certain cargo was prevented from entering MDCs. Since fluorescent markers were only on the Tcd2 cargo protein, it was unknown whether MDCs were still being formed with just Tom70. For this reason, Tom70 was tagged with both GFP and UL36, which confirmed ubiquitination as required for MDC formation (Fig. 5).
The inhibition of MDCs in yeast strains expressing Tom70-GFP-UL36 indicates that UL36 blocks MDC formation rather than simply blocking cargo from entering MDCs. Future research should focus on if the observed ubiquitin requirement is specific to Tom70 or if it is the overall presence of ubiquitin that is necessary for MDC formation. Ongoing experiments involve inducing MDCs by rapamycin in strains expressing Tom20-UL36. Tom20, another import receptor on the OMM, is unaffiliated with MDC formation, leading us to expect that MDCs will not be blocked. This would suggest that it is not the overall presence of UL36 but rather the deubiquitination action of UL36 on or near Tom70 that is blocking MDCs. Only one replicate of the study has been performed, so additional experiments are needed.
To further determine the specificity of ubiquitination in MDC formation and cargo loading, we are performing additional MDC experiments in strains expressing Tcd2-GFP-UL36 and Tom70- mCherry. Based on the hypothesis that ubiquitination is specific to Tom70 for MDC formation, it is expected that Tcd2-GFP-UL36 will not inhibit Tcd2-positive MDCs or Tom70-positive MDCs, which would confirm UL36 to block MDCs due to the inhibition of Tom70’s ubiquitination. Together, the Tom70-GFP-UL36, Tom20-UL36, and Tcd2-GFP-UL36 experiments would confirm Tom70-specific ubiquitination to be a requirement of MDC formation. Results other than those expected may suggest that UL36 prevents ubiquitination of all proteins or MDC-affiliated proteins, which would require further investigation.
Materials and Methods
SCM4-OE Strain Construction
To create yeast strains that overexpress SCM4, we integrated a linearized plasmid containing SCM4 under a GPD promoter for constitutive overexpression (OE) into Chromosome 1 of Saccharomyces cerevisiae. An empty vector negative control was made by inserting a linearized plasmid with no gene insert into Chromosome 1. Plasmids for both OE and EV strains were integrated into yeast strain that expressed Tcd2-GFP (for MDC visualization), Tim50-mCherry (for mitochondrial tubule visualization), and Tom70-UL36 (DUB fusion protein). Colony PCR and selective growth confirmed the correct integration into Chromosome 1.
Tom70-GFP-UL36 Strain Construction
To create a yeast strain expressing Tom70 C-terminally tagged with GFP-UL36 (Tom70-GFP-UL36), a DNA fragment containing GFP-UL36-3xHA and flanking homology to the 3’ end of TOM70 was amplified by DNA PCR using Phusion Polymerase. The PCR product was then transformed into a yeast strain that expressed Tim50-mCherry. Tagging of Tom70 was verified by observing the mitochondrial membrane-localized GFP by microscopy and the detection of full-size Tom70-GFP- UL36-3xHA by western blot with Tom70 or HA antibody.
Yeast Cell Culturing
Yeast cells were grown shaking at 30°C overnight, prior to experiments, in YPAD (1% yeast extract, 2% peptone, 0.005% adenine, and 2% glucose) to a density of 0.3-0.8 OD600. Drug treatments used and their respective concentrations were the following: cycloheximide (1 µg/mL), rapamycin (200 nM), and auxin (1 mM).
MDC Assays
Post the overnight growth to log phase (0.3-0.8 OD600), cell cultures were treated with the indicated drug or dimethyl sulfoxide (DMSO) as a control for their respective durations. Live cells were prepared for imaging by harvesting via centrifuge, washing out the culture media, and then resuspending cells in a buffer solution (5% glucose, 10mM HEPES pH 7.6). Optical z-sections of live yeast cells were acquired with a ZEISS Axio Imager M2, 63x oil immersion objective. Widefield images were acquired via ZEN and processed using Fiji. MDCs were then quantified in ZEN.
Protein Lysate Preparation and Immunoblotting
For Western blot analysis of Tom70-GFP-UL36-3xHA, yeast cells were first revived from frozen stocks and incubated at 30°C for 2 days. Using the grown single colonies, a 3mL culture was grown overnight for 16-17 hours at 30°C and then back diluted (0.5µL into 50mL media) for 16-17 hours at 30°C until cells reached log phase (0.3-0.8 OD600). 2 ODs of log phase cells were harvested via centrifuge, washed with distilled water, and resuspended in ice cold lysis buffer (0.2 M NaOH, 0.2% β-mercaptoethanol). Following 10 minutes of incubation on ice, Trichloroacetic Acid (TCA) was added at a final concentration of 10% and samples were incubated on ice for 10 minutes. Samples were then spun for 10 minutes at 13,000 rpm to create a pellet, which was then resuspended in 60µL 2x SDS lysis buffer plus 15µL 1M Tris base (pH 11), then incubated at 50°C for 30 minutes, briefly placed on ice, and centrifuged for 1 minute at 13,000 rpm. 4µL of samples were loaded into 4-12% BIS-TRIS wedge well gels in MES running buffer and then run at 120V for 1-2 hours. Proteins were transferred to nitrocellulose membranes via semi-dry transfer. Post transfer, the membranes were rinsed with water and then stained with Ponceau S. An image of the Ponceau stain was then taken on a BioRad ChemiDoc imager. 5% milk in PBS + 0.05% Tween-20 (PBST) was added to the blotting boxes for 1 hour of rocking at room temperature. Membranes were incubated in their appropriate primary antibody solutions, rocking for 1 hour at room temperature. After being washed 3 times for 10 minutes in PBST, membranes were incubated in their appropriate secondary antibody solutions, rocking for 1 hour at room temperature. Post being washed 3 times for 10 minutes in PBST, membranes were developed in Pierce Dura Extended Duration ECL solution and imaged using a BioRad ChemiDoc imager.
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