A new study from a team of Canadian and German researchers has uncovered never-before-seen details about what happens to proteasomes, the cell’s garbage disposal system, when there is not enough energy.
The findings, which were published in the journal Cell, show that when cells are starved, proteasomes become arranged into crystal-like structures to form proteasome storage granules (PSGs). This discovery upends previous assumptions about the liquid droplet-like nature of PSGs and represents a technical feat in visualizing cellular compartments in their native states.
Cells organize their molecules in distinct functional areas known as organelles. While textbooks usually refer to membrane-bound organelles like mitochondria and cell nuclei, recent studies have also revealed organelles, such as PSGs or stress granules, that are not enclosed by membranes. In the past, these membrane-less organelles were only visible as droplets using a fluorescence microscope.
“As PSGs coexist with multiple membrane-less organelles during cellular stress, we set out to determine how their molecular architecture enables the selective confinement of proteasomes within the cellular environment,” says Cordula Enenkel, the study’s last author and a former professor of biochemistry at the University of Toronto’s Temerty Faculty of Medicine.
Co-led by Enenkel and Wolfgang Baumeister and Brenda Schulman from the Max Planck Institute of Biochemistry, the researchers used yeast to study the function and structure of PSGs.
“We subjected yeast cells to energy stress by depriving them of glucose or blocking mitochondrial ATP production. This created a scarcity of ATP, the cells’ energy source,” says Enenkel, who is an expert in proteasome biology. “In response, the cells shut down their energy-intensive proteasomes by arranging them into PSGs. This revealed a fascinating cellular strategy to cope with metabolic stress.”
The research team, which also included U of T biochemistry professor Oliver Ernst, first tried to study the structure of PSGs in a test tube, but failed because the granules kept disintegrating. They next turned to cryo-ET, a high-resolution microscopy technique pioneered by Baumeister that allows researchers to see the 3D structures of protein complexes in their natural cellular environments.
Using cryo-ET, the researchers could visualize the PSGs at a resolution of 0.9 nanometers, which revealed structural details that were previously impossible to see. They observed for the first time that in the granules, individual proteasomes were arranged into precise, repetitive structures.
The researchers also showed that proteasomes assemble into PSGs by first forming trimers — three proteasomes bound together — which then stack into fibres. In the final step, bundles of fibres came together to form the crystal-like structures.
A proteasome is a large protein complex that works as an energy-powered protein shredder to eliminate unneeded or damaged proteins by chopping them into small pieces. When certain nutrients or other sources of cellular energy are too low, the locations of proteasomes inside cells are visibly altered.
In yeast cells, the proteasomes in the PSGs remain fully assembled but inactive, so they do not waste energy during energy shortages. The PSGs are stable under energy-limited conditions, but the PSGs can also be shattered, which allows rapid reactivation of the proteasomes when a source of energy becomes available. When the researchers supplied glucose to the starved cells, the proteasomes rapidly resumed their normal functions.
“The discovery provides a new view of membrane-less organelle formation, through contacts between well-structured regions of molecular machines,” says Schulman. “This allows PSGs to safely store proteasomes — which are energetically costly to make and essential — in reserve, ready for deployment when cellular conditions improve.”
“Studying structures inside of cells, like proteasomes in their native environment, fundamentally changes our understanding of cellular organization and function,” says Baumeister.
This research was supported by the Canadian Institute for Advanced Research (CIFAR), Canadian Institutes of Health Research, Deutsche Forschungsgemeinschaft (German Research Foundation), Ernst Jung Foundation, European Union and Max Planck Society.