The cell is a dynamic environment. Molecules are constantly entering and leaving, and proteins are always being synthesized, regulated, and transported. Yet somewhere in all this constant movement of materials, the proteins that support the activity and structure of the cell may become damaged, broken, or worn, and so must be degraded.
How does the cell know when a protein has reached the end of its productivity, and how does it go about degrading this one specific, broken protein, in the midst of countless others? Jeff R. Jorgensen, a graduate researcher in the Scott D. Emr lab, Department of Molecular Biology and Genetics and director of the Weill Institute for Cell Biology, conducts research, hoping to answer these questions.
How a Protein Is Marked for Degradation, Then Travels across the Cell to Its Destiny
“Proteins don’t last forever,” Jorgensen explains. “Cells need some sort of mechanism to degrade broken proteins so that they can be turned over.” The Emr lab specifically focuses on how plasma membrane proteins, the ones that separate the interior of the cell from its surroundings, are identified and subsequently broken down. The process of degradation requires the membrane protein to leave the membrane and traffic through the cell in order to reach the structure that degrades it. In mammals, this structure is known as the lysosome, but in yeast cells (the model organism that Jorgensen works with) it is the lumen of the vacuole.
First, a protein named ubiquitin tags the membrane protein, marking it in order to identify it for degradation (a process known as ubiquitination). This triggers an endocytosis event, in which the membrane protein is brought into the cell, contained in a small membrane sac called a vesicle. The vesicle then fuses with an endosome (another sac-like structure), delivering the plasma membrane protein to its limiting membrane.
As the endosome matures, a set of protein complexes called the ESCRT machinery recognizes the ubiquitinated proteins and pinches them off into smaller vesicles within the endosome. The resulting structure is known as a multivesicular body, because it consists of one large endosome that contains multiple vesicles. This multivesicular body is trafficked to the vacuole in the yeast cell (the lysosome in mammalian cells), where it then fuses to the vacuole membrane. The endosome becomes incorporated into the vacuole membrane, while the vesicles are deposited into the vacuole lumen, where they (along with the marked membrane proteins inside them) are degraded.
Hypothesizing about How Proteins of Yeast Cells are Degraded
However, plasma membrane protein degradation is only half of what Jorgensen and the Emr lab are interested in. They also study how the vacuole membrane proteins themselves are degraded. “The vacuole is thought of as a terminal destination. It’s where things go in order to be broken down. So how do the vacuole membrane proteins—the proteins that allow specific molecules to traffic between the inside of the vacuole from the rest of the cell—get brought into the vacuole and turned over?” In other words, how do the proteins of the terminal destination, themselves, get trafficked to the terminal destination?
“The vacuole is thought of as a terminal destination. It’s where things go in order to be broken down.”
Jorgensen explains that he originally had two hypotheses as to how vacuole member proteins were able to enter the vacuole lumen for degradation without compromising the structure of the vacuole itself. The first was a recycling model. The recycling model functions similarly to the way in which integral plasma membrane proteins are broken down: First, they are marked (ubiquitinated); then shuttled off the vacuole membrane in an endosome; the ESCRT machinery forms a multivesicular body; then fuses back with the vacuole membrane to deposit the vacuole membrane protein into the lumen of the vacuole.
The second hypothesis is a direct invagination model. Instead of the vacuole membrane protein being marked, shuttled off the vacuole membrane into the cytosol, and returned to the vacuole—which is what the recycling model posits—in the direct invagination model, the ubiquitinated protein would travel directly into the lumen of the vacuole.
The Experiments—Testing the Hypotheses on the Yeast Protein, Ypq1
To test these hypotheses, Jorgensen’s experiments focused on how a specific vacuole membrane protein, known as Ypq1, was trafficked. He used a genetic approach to identify genes that are required for Ypq1 trafficking. Ypq1 is normally ubiquitinated and transported into the vacuole lumen in order to be degraded in the absence of lysine. Jorgensen, however, set up a genetic selection for cells that were unable to degrade Ypq1.
There were three general mutant classes. The first class was one in which ubiquitination was blocked, so Ypq1 could not be marked for degradation. In these mutants, Jorgensen expected to find that Ypq1 remained on the vacuole membrane, never being degraded because it was not able to be marked. In the second class, the ESCRT machinery was mutated, preventing the formation of multivesicular bodies. Jorgensen predicted that these mutants would allow ubiquitination of Ypq1 and traffic to endosomes, but that a multivesicular body could not be formed, and Ypq1 would be stuck on the endosome. In the third class, the fusion between the endosome and the vacuole membrane was disrupted. A multivesicular body containing Ypq1 could form, but it could not fuse to the vacuole membrane, so the Ypq1 would be stuck inside the multivesicular body.
Jorgensen developed a technique to bypass the first class of mutants, in which ubiquitination occurred even in the presence of the mutation. What he found then was that the degradation of Ypq1 proceeded normally in this class of mutants. He further applied this technique to the other two classes of mutants to ensure that Ypq1 was always ubiquitinated and therefore marked for degradation.
What was surprising, however, was that in the ESCRT machinery, mutants Ypq1 never left the vacuole membrane, even though it was ubiquitinated. Ypq1 was never removed to an endosome. In fact, the endosome-vacuole fusion mutants functioned normally under these circumstances. This information works against the recycling model, because it implies that the formation of an endosome and multivesicular body is not necessary for the degradation of Ypq1. In ESCRT mutants, Ypq1 was not found on endosomes; it never even left the vacuole membrane. Instead, it seems more likely that the direct invagination model is correct, that Ypq1 is trafficked from the vacuole membrane into the lumen of the vacuole, and that the ESCRT machinery works directly at the vacuole membrane in order to transport ubiquitinated membrane proteins into the lumen.
The Research—Leading to an Understanding of Protein Quality Control in Cells
“It takes at least 20 minutes to explain my research to an outsider,” Jorgensen laughs, “but this research allows us to understand protein quality control in cells and how a cell maintains homeostasis.”
Jorgensen’s work in the Emr lab has also marked changes in the type of research he performs. “When I applied, I was looking to do something similar to what I had done—developmental biology, primarily focused on mice models and chicken embryos as model systems. But I’ve found that the speed at which you can genetically manipulate yeast is amazing. My favorite part of my research has been just to set up genetic selections that allow yeast to show us what is important. I’m letting the yeast guide me and what I’m studying, instead of allowing my own biases—what I want to see—guide me.”