First person – Fumiya Okawa, Yutaro Hama and Sidi Zhang

Sidi Zhang, Yutaro Hama and Fumiya Okawa

How would you explain the main findings of your paper in lay terms?

F.O., Y.H. and S.Z.: Cells survive by continually synthesizing, destroying and rebuilding themselves. One destruction pathway is autophagy, during which autophagosomes, structures that eat cellular components and break them down on a large scale, are produced. Autophagosome formation is the most important step that characterizes autophagy, but how it occurs is not fully understood. Among the most enigmatic aspects of autophagosome formation is the requirement for two proteins, TMEM41B and VMP1, which are evolutionarily related to each other. Both TMEM41B and VMP1 are found in the endoplasmic reticulum, an intracellular organelle distinct from the autophagosome, but their specific functions are largely unknown. Proteins related to TMEM41B and VMP1 are present in a wide range of organisms, including bacteria. In this study, we focused on the evolutionary relationship between these proteins, and tried to predict the functions of TMEM41B and VMP1 by taking a hint from their evolutionary histories. First, we analyzed the evolutionary relationship between the relatives of TMEM41B and VMP1 in detail, and found that they form the DedA superfamily, with TMEM41B and VMP1 included in separate families. Furthermore, we predicted the three-dimensional structure of the DedA domain common to the DedA superfamily based on evolutionary information, and biochemical experiments confirmed that the prediction was largely correct. Since a structure similar to the DedA domain is found in membrane proteins that transport small molecules across membranes, we propose that TMEM41B and VMP1 may transport some substances required for autophagosome formation from the endoplasmic reticulum. Our findings provide a basis for clarifying not only the molecular mechanism of autophagy, but also its evolutionary origin.

Were there any specific challenges associated with this project? If so, how did you overcome them?

F.O., Y.H. and S.Z.: We wanted to accurately describe the phylogenetic relationships among the VTT domain-containing proteins (now the DedA superfamily). However, since classic phylogenetic analysis methods are based on comparing primary sequences with some degree of homology, the analysis of remote homologs with low sequence similarities has been severely limited. This problem was solved with the introduction of ‘graph splitting’, a new phylogenetic analysis method for remote homologs, developed in 2020. Graph splitting creates a phylogenetic tree by pairwise sequence comparison and therefore relaxes the requirement for sequence homology. Using this method, we succeeded in reconstructing a convincing phylogenetic tree. From the data, we found that the VTT domain-containing proteins have divided into four subgroups since they separated from the ancestor, and we named the whole group the DedA superfamily. This finding is the starting point of this paper.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

F.O., Y.H. and S.Z.: Before we started this study, we noticed two things from the sequence analysis of the VTT domain (now the DedA domain). One is that it is difficult to predict transmembrane helices in the VTT domain (this is why it was believed that VMP1 has six transmembrane helices instead of eight!), and the other is that there are conserved proline residues even though the primary sequences of the VTT domain have low homology. These questions were resolved in one fell swoop when a predicted structure containing two facing reentrant loops was obtained. In other words, the difficulty in predicting transmembrane helices was due to the low hydrophobicity of the reentrant loop, and the conserved prolines locate in the folded part of the reentrant loop. However, when the predicted structure was first obtained, we were a bit skeptical. But we were subsequently able to verify the topology using the substituted cysteine accessibility method (SCAM). We were impressed by the fact that this data confirmed the structure predicted in silico by biochemical methods, and truly connected dry and wet biology.

Why did you choose Journal of Cell Science for your paper?

F.O., Y.H. and S.Z.: We feel that Journal of Cell Science publishes very solid and essential content covering a wide range of topics in cell biology, and we refer to it a lot in our research. Many cell biologists read it regularly, so it can help our paper reach a wider audience. Also, we think the cool design is one of its attractive features.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

F.O., Y.H. and S.Z.: Yes, the guidance and support from our PI, Dr Noboru Mizushima, was essential for us. He recognized our different backgrounds and individual strengths and brought us together as a team, encouraging our multidisciplinary research. He has lots of great ideas, and his passion and dedication to science is contagious. We feel that we have really broadened our horizons and become better researchers under his guidance.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

F.O.: When I was a child, I thought we knew everything there was to know about the world. In junior high school, I finally realized that I was wrong, because my science teacher told me that one of my questions was still unsolved. I was relieved and excited at the same time. My future goal is to conduct research that will lead to cures for diseases. However, I think that there are many things that we don't understand about the mechanisms of living things. When things don't go as expected, the process of solving them through trial and error is a successful experience, no matter how trivial it may seem. I would like to try to ask important questions and proceed in this exciting field through trial and error.

Y.H.: Through the activities in my high school science club, I learned the fun of experimental science, where you can solve problems with your own ingenuity and teamwork, and that research is open to society. My experience participating in a state-sponsored program in high school led me to pursue molecular biology. I went to university to experience the most advanced molecular biology experiments. In an experiment where E. coli was transformed to express GFP, I was very strongly moved when I saw the colony of E. coli glowing green under the excitation light, because I visually recognized the fact that I had modified life with my own hands. I then decided to major in life science, hoping to do research using this technology. Even now, when I see a cell expressing a GFP-tagged recombinant protein glowing green, I remember how I felt at that time. I am very grateful to my science club advisor at the time, Mr. T.O., who allowed me to have these experiences.

S.Z.: I really like animals and grew up with many pets. Observing and interacting with them sparked my initial interest in biology. In college, I became particularly interested in evolutionary biology. Putting things under the lens of evolution adds a historical perspective and helps me appreciate the grandeur of life more, which continues to motivate me as a researcher.

Who are your role models in science? Why?

F.O.: Dr. F. K. (I am sorry for the initials) is one of my mentors. Although he is already over 70 years old, he is still active in research. Whenever I meet him, he tells me about the progress of his research. Recently, I heard that he has been conducting experiments on his days off as well. He is one of my role models. I know it is not an easy thing to do, but I hope to be involved in research for a long time.

Y.H.: For me, my colleagues, supervisors and all the researchers around me are role models. Research is creative work, and in order to come up with new concepts, it is important to sharpen my own sensibilities and gain new perspectives. What makes this possible, I believe, is to value my own individuality while constantly incorporating new stimuli into it and ‘evolving’. In addition, people are multifaceted, with different personalities, abilities and strengths. It would be a shame to overlook certain aspects in people and not use them as role models. For these reasons, I would like to keep learning from the researchers around me.

S.Z.: I am lucky that I met a lot of great supervisors. They taught me the importance of curiosity and perseverance in science and helped me grow as a person. They have brought me to where I am and will continue to inspire me moving forward.

What's next for you?

Y.H.: I have just completed my PhD while doing this interview! I plan to continue my research on autophagy as a postdoctoral fellow, enjoying my research and working to elucidate the molecular mechanisms and evolutionary origins of autophagy.

S.Z.: I want to continue research. Life has this beautiful complexity, and many processes are still elusive; I want to be part of the endeavor to uncover its mysteries.

Tell us something interesting about yourself that wouldn't be on your CV

Y.H.: I have a hobby of protein sequence analysis. On days when my experiments don't go well, or on nights when I can't sleep, I play with some protein sequence. It is so much fun to see the true face of a protein through its amino acid sequence, and it supplies me with the energy to face experiments. This is a very fun hobby that can lead to unexpected discoveries.

S.Z.: When I am not working, I like to watch figure skating tournaments. The jumps, spins and the carefully choreographed performances to music are always very enjoyable, and they remind me that behind every shiny and seemingly effortless moment are tons of hard work and team effort.