As a first-year graduate student in the laboratory of Bonnie Bartel, PhD, professor of biosciences at Rice University, Zachary Wright, PhD, now a postdoctoral research associate in Bartel’s lab, identified hidden compartments within peroxisomes, an essential part of the sub-cellular machinery found in all eukaryotes, from yeasts to humans.
Peroxisomes are linked to severe metabolic disorders, neurodegeneration, obesity, cancer, and aging-related disorders. The basic structure of peroxisomes has long been held to consist of a dense granular protein-filled matrix surrounded by a single lipid-bilayer membrane. Although much remains unknown about peroxisomes, they play key roles in β-oxidation of long-chain fatty acids and reduction of reactive oxygen species.
Published in Nature Communications, these findings were initially surprising to Bartel, who questioned the validity of these unexpected internal compartments in peroxisomes, wondering whether they could be experimental artifacts. “I never thought Zach did anything wrong, but I didn’t think it was real,” said Bartel. “If this was really happening, somebody would have already noticed it,” she recalled thinking.
To address his advisor’s incredulity, Wright set about checking his instruments, replicating his experiments, gathering more data, and combing through earlier studies that reported on the structure of peroxisomes.
Wright discovered reports of similar internal compartments in peroxisomes in studies from the ‘60s and ‘70s, that used transmission electron microscopy which fell out of favor with the advent of confocal microscopy. “They had observed similar inner compartments and just didn’t understand them,” he said. “And that idea was just lost.”
Wright also used confocal microscopy in his studies, but he used brighter fluorescent tags that allowed high resolution imaging. Another factor that was a key advantage in Wright’s studies was that instead of using yeast or mammalian cells as his model of choice, he studied peroxisomes in Arabidopsis thaliana seedlings where peroxisomes are up to 100 times larger (~1–2 μm).
Peroxisomes in Arabidopsis seedlings grow larger because their seeds use fat and oil as their primary source of energy before leaflets sprout and photosynthesis kicks in. Peroxisomes go into overdrive, burning fatty acids to provide adequate sustenance for the seedling, expanding manifold in the process.
“Bright fluorescent proteins, in combination with much bigger peroxisomes in Arabidopsis, made it extremely apparent, and much easier, to see this,” said Wright.
Some eukaryotic mutations lead to engorged and less densely packed peroxisomes, but these have not been used in characterizing the basic internal structure of peroxisomes as this phenotype results from known mutations.
The inner membrane-bound compartments in Arabidopsis peroxisomes, the authors noted, are formed through the bending and budding of its outer membrane, using a molecular machinery called endosomal sorting complexes required for transport (ESCRT).
The internal compartments or intraluminal vesicles accumulate over time and do not form normally when β-oxidation is disrupted. Fatty acids are imported into peroxisomes for oxidation by the ABC transporter PXA1. The authors showed peroxisomes in several mutants of PXA1 lack inner compartments and present the classical morphology of a protein-filled sac with a single lumen.
Although Wright is not sure of the reason behind peroxisomal compartmentalization, he has a working hypothesis. “When you’re talking about things like beta-oxidation, or metabolism of fats, you get to the point that the molecules don’t want to be in water anymore,” said Wright. Wright suspects the internal membranes in peroxisomes solubilize and mobilize water-insoluble metabolites, allowing better access to luminal enzymes.
Bartel believes this elucidation of the ultrastructure of peroxisomes will provide a new context for investigating peroxisomal disorders. “This work could give us a way to understand some of the symptoms, and potentially to investigate the biochemistry that’s causing them.”
Bartel added, “This discovery requires us to rethink everything we know about peroxisomes.”
