63 Analyzing the role of Tom70 in supporting MDC formation: A Research Reflection

Faculty Mentor: Zachary Wilson (Biochemistry, University of Utah)

A research reflection

Upon my acceptance into SPUR, the immediate excitement from family, friends, and even faculty was followed by a questioning look, all questioning the same thing, “Have you ever even been to Utah?” This question made sense, after all, being born and raised in Washington State, there has been little to no time outside the state without my friends or family for comfort. So, each time my answer was a chuckle laced with nervousness, excitement, and even the tiniest bit of fear followed by a small “No.” I had a small idea of what kind of research I was getting into, knowing I would be studying mitochondria in the model organism S. Cerevisiae, cool right? The powerhouse of the cell! That in and of itself was a change, having only studied Pacific Madrone genetics and the DNA repair pathway of Tetrahymena thermophila, switching to a different organism with research heavy in biochemistry (which I have to admit, I wasn’t stellar at) was a little nerve wracking, but even more exciting.

So, I made my way here to Salt Lake City, curious what my three new roommates would be like, if we would get along, and the same about my mentor. I had heard good things about Zach from a professor I worked closely with back home, so I had an idea of what to expect, but as most people know, what you expect, and reality can be completely different. Luckily for me, what I heard was true! Cue the automatic relief. This relief continued as I met the other members of the Hughes lab, everyone being genuinely friendly and open. The kindness balanced out my nerves, and the sudden weight of expectation that I was feeling seemed a little lighter. At least until I saw the lab. Now, being from a smaller university our labs are still amazing, but this was on a completely different level. About five times the size of my lab back home, and filled with tools and machines I didn’t even know existed, the pressure started increasing. I doubted if my work would be up to the caliber of what was expected from this lab. So of course in response, my determination to be a welcome addition to the lab increased tenfold.

Zach and I spent the first couple of weeks just introducing me to the project, going into the background of what research the lab had done previously, and how my project would fit in and contribute to others projects. At first, this was information overload, papers upon papers, wrapping my head around the fact that yes, sometimes the incubation rooms would smell like bread, and learning to get the timing right for yeast and bacterial cultures. But eventually I got the hang of it, and even got all the way through my project!

Now, I’ve been mentioning “the project” but haven’t actually explained what I dedicated my summer to! The Hughes lab focuses around how the organization of cellular proteins and metabolites drives aging and disease. As I’ve said, my project was centered around the mitochondria, which perform an essential role in numerous metabolic pathways. Defects in mitochondrial function are associated with the progression of several age-related neurodegenerative diseases. The Hughes lab recently discovered a mitochondrial degradation pathway, called mitochondrial-derived-compartments (MDCs) that is induced in response to several cellular stressors, as well as in old-aged cells. The lab has shown that MDCs selectively remove a subset of membrane proteins from the outer mitochondrial membrane and that failure to form these structures exacerbates mitochondrial dysfunction, suggesting that the MDC pathway protects mitochondria in times of stress. The formation of MDCs requires the conserved import receptor Tom70, an outer mitochondrial membrane protein that facilitates the import of hydrophobic proteins into the mitochondria and acts as a cochaperone that assists in reducing the proteotoxicity of aggregation-prone proteins. Currently, the role that Tom70 plays in MDC formation is unknown. This is where my part came in. To analyze the function of Tom70 in MDC formation, we used the budding yeast, Saccharomyces cerevisiae, to create genetically mutated strains that expressed either truncated versions of the Tom70 protein, which systematically removed functional domains, or expressed Tom70 mutations that removed binding to cytosolic chaperones. The results determined that the majority of Tom70 mutations analyzed were unable to rescue the temperature sensitivity of a tom70Δ strain and were also unable to support MDC formation. Likely, many of the HA-tagged truncations destabilized the Tom70 protein, and therefore disrupted MDC formation. The plasmids created were found to more stably express Tom70 mutants and the results suggest that Tom70 binding to chaperones is not required for MDC formation.

Overall my research this summer provided an important first step in elucidating key features of Tom70 that are required for MDC formation. Doing this work allowed me to get experience with things I’ve never done, western blots, sequencing samples to ensure truncations were being made, designing primers, doing Gibson Assemblies to create the plasmids with single point mutations and bigger mutations like taking out the whole chaperone binding domain of Tom70. This summer helped me grow both as a researcher, and in general. I learned how to be comfortable being uncomfortable, being in a different field of research this summer felt like one big learning curve. However not once did I feel like it was a bad thing, or that I wouldn’t be able to complete the summer. If anything, being exposed to new research, techniques, and other aspects helped me adapt more to what I was learning. I also learned a lot about my own love for doing research. It solidified my desire to pursue a Ph.D. and start doing my own projects, no matter how far in the future that may be.

Now, with less than a week left and my project wrapped up, leaving will be bittersweet. I will desperately miss doing research full time in this awesome lab, full of amazing people. People who I asked for advice and help with my project, but also went to the movies with, tried to walk a tightrope with, and commiserated about the heat with. I feel very fortunate to have gotten Zach as a mentor because he made sure that with every experiment or test I knew what the purpose was, and what the result meant. He checked in with me everyday, was patient with my repeated questions, and always made sure I was doing okay. The other lab members were just as fantastic, answering my random questions, giving me memes to hang up on my workbench, and generally just being friendly and approachable in a way I would not have expected from graduate students and postdocs. I will also miss my roommates, while we all worked hard we still made time for family dinners, trips to the water park and various lakes, pizza and movie nights, late night drives, and lots of fashion shows. Overall, my time here in Salt Lake City has been amazing and while it somewhat hurts to leave so soon, and trips back will definitely be on the horizon, I am looking forward to going home. Thank you for this opportunity.

References for my research

Hughes, A. L., et al., (2016). Selective sorting and destruction of mitochondrial membrane proteins in aged yeast. Elife, 5, e13943.

Backes, S., et al., (2021). The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress. Cell reports, 35(1).

Schuler, M. H., et al., (2021). Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress. Molecular Cell, 81(18), 3786-3802.

English, A. M., et al., (2020). ER–mitochondria contacts promote mitochondrial-derived compartment biogenesis. Journal of Cell Biology, 219(12).