In a new publication in The Plant Cell — “Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus” — authors Philip Day, Steven Theg, and Kentaro Inoue, all at University of California, Davis, determined how β-barrel proteins are sorted to the correct location in plant chloroplast envelopes, which have two membranes. Chloroplasts, which are responsible for photosynthesis in plants, evolved about a billion years ago from an ancient endosymbiotic relationship between a cyanobacteria species and a eukaryotic cell.

This story of how a graduate student and his two major professors made a significant find didn’t follow a straightforward path. It ended up being a testament to great research, resilience, perseverance, and keen mentoring.

“In both my labs at UC Davis — Steven Theg’s lab, and Kentaro Inoue’s lab — everybody was really interested in how proteins are correctly sorted to different places within the chloroplast,” said Philip Day, who co-published this paper just as he was finishing his Ph.D. at UC Davis. Theg and Inoue both served as Day’s major professors.

“The chloroplast is a complex structure,” added Day. “It has two envelope membranes, and a thylakoid membrane, and in between all of these membranes there are aqueous areas. To get the proteins to their specified locations, there are a lot of signals and machinery needed to make this happen, which is what we’re trying to understand.”

Day’s work was about a very specific type of protein — β-barrel proteins, which are inserted into biological membranes. This type of protein is found only in a few other types of organism membranes, including bacteria outer membranes and mitochondria outer membranes. He and his major professors had to figure out how the β-barrels, which are transmembrane proteins, are specifically sorted to the chloroplast outer membrane.

Switching professors midway through a Ph.D. program

Philip Day started as a graduate student in the Plant Biology Graduate Group at UC Davis in 2012, and joined Kentaro Inoue’s research lab in the Department of Plant Sciences in 2013. Day was making headway in his research, when Kentaro Inoue, his major professor who was an avid bicyclist and known for his sense of humor, was tragically killed in a bike accident in 2016.

The loss of Inoue, who was originally from Japan, was felt across UC Davis and by his many global colleagues. Within Inoue’s lab, three graduate students (Philip Day, Lucas Mckinnon, and Laura Klasek), all working on chloroplasts, were suddenly adrift following the loss of their major professor.

Steven Theg, a professor in the Department of Plant Biology at UC Davis, who also works on protein translocation across chloroplast membranes, stepped in to see how he could help with Inoue’s graduate students and their research.

“I already knew the research that Philip was doing with Kentaro,” said Theg. “Through our shared interests we knew each other’s students and research quite well. At the time, I already had a full load of graduate students, but when Kentaro passed away so unexpectedly, it made complete sense for them to join my group.

“Not only were they doing important work, but this was a critical time during their graduate studies. I hoped that if his students joined my group, we could continue Kentaro’s research with a minimum of interruption. This would be Kentaro’s legacy."

It was only possible to do this with the commitment from Plant Sciences chairs Joe DiTomaso, and later, Gail Taylor, along with Dee Madderra, to continue their departmental student support until they graduated.

Day notes that the switch to Steve Theg as a major professor went as smoothly as could be expected at a time of sadness and upheaval, and that he will forever be grateful to Theg for helping him and his two graduate student colleagues continue their original research.

Why chloroplasts?

“The evolutionary history of chloroplasts is one area I wanted to explore when I started graduate school at UC Davis,” said Day. “As an undergrad at University of Nebraska, I learned about this theory of endosymbiosis, and about how both chloroplasts and mitochondria arose from bacteria that basically took up residence inside of a eukaryotic cell. That just blew my mind, and I set my sights on doing graduate research on chloroplasts.”

He stuck with his plan and succeeded quite well.

“What I’m proudest about in this research is my work on the OEP80 protein,” said Day. “It uses a transit peptide, which is uncommon for outer membrane proteins and was unexpected. That was one of the first discoveries that I made, and it’s included in this paper.”

In imagining how his current work on β-barrel proteins might one day have application, Day said that just about everything we learn in biology could have different sorts of application, or uses beyond our present understanding.

“β-barrel proteins, found on the outside of bacteria, might be good targets for vaccine production,” said Day. “In chloroplasts, people can express bacterial proteins, which they can use to make vaccines, so they don’t have to make a vaccine using the pathogen. They can take the protein and accumulate it in plant cells.”

Looking ahead, Day says that a possible application of his research would be to produce bacterial or pathogen β-barrels in plant cells. 

Moving forward

Philip Day left UC Davis this summer, after completing his Ph.D. with Professor Theg. He moved north to Pullman, Washington to take a postdoctoral research position with Professor Henning Kunz at Washington State University. Day will continue working on chloroplast research there, moving beyond his research at UC Davis.

“What I did in my Ph.D. was mostly about how these proteins are sorted to the correct location,” said Day. “For my postdoc, I’ll be working on the chloroplast and the chloroplast envelope which has two membranes, and how ions and other solutes are transported through the envelopes.

“During my graduate studies at UC Davis, I became a better scientist and gained a passion for studying plant cells and plant biochemistry due to the mentoring of both professors, Kentaro Inoue and Steve Theg. This experience opened the path for me to continue researching chloroplasts and grow as a scientist.”

Added Professor Theg, “With his Ph.D. work, Philip has added important missing information about chloroplasts, not only concerning their biogenesis and maintenance, but their evolution as well. I expect his paper will be widely read and cited by our colleagues around the world who are interested in this essential plant organelle.”