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The nitty-gritty of peroxisome biology is becoming clearer, thanks to a resurgence of interest in the organelle by researchers such as Richard Rachubinski (left), working with yeast, and Bonnie Bartel, working with plants (see sidebar).
For about eight years, starting in the mid-1990s, they took genes that Kunau and others had found were needed to form peroxisomes in yeast and searched for equivalent human genes in a sequence database. When they got a match, they isolated the candidate human gene and added it to one of the Moser's cultured skin cell lines to see if it restored working peroxisomes. Nine did. Today, mutations in 13 different genes are known to cause human peroxisome biogenesis diseases. Those genes provided a parts list for the human peroxisome, albeit an incomplete one. Fundamental questions remained.
One such question was: how is the organelle formed? Dogma had it that new peroxisomes formed when existing ones divided—the same way that mitochondria reproduce. But cell biologist Richard Rachubinski, an HHMI international research scholar at Canada's University of Alberta, believed otherwise. His team kept finding evidence that peroxisomes were produced by the endoplasmic reticulum (ER), a network of flattened sacs resembling a stack of pancakes that helps the cell make membranes and package proteins for shipment out of the cell. In yeast studies, they found one type of immature peroxisome that seemed to be budding from the ER. They noted that two peroxisomal proteins were covered with sugars that are attached to protein only when they move through the ER.
Others in the field were skeptical. When his team submitted papers to journals, reviewers demanded control after control, so many that one paper ballooned to more than 60 figures. “We got roasted, constantly,” Rachubinski recalls.
The debate raged for more than a decade. Then, in 2005, Henk Tabak, a cell biologist at the University of Utrecht, the Netherlands, created a hybrid protein—half peroxisomal membrane protein called Pex3, and half green fluorescent protein from jellyfish. In cells with the hybrid, green spots clustered first on the membrane of the ER. The green clusters would then bud off as a vesicle and mature into a normal (albeit green) peroxisome. The green peroxisomes formed only in the presence of Pex19, a protein known to be required for peroxisome assembly, the researchers reported in Cell. In an accompanying commentary article, Schekman recapped the popular view that peroxisomes were autonomous and then added that, “the authors of cell biology textbooks may wish to reconsider this view when they write their next edition.”
Later that year, Rachubinski's group reinforced the case for peroxisomes emanating from the ER. First they created a hybrid protein similar to Tabak's with a part of Pex3 attached to green fluorescent protein. The hybrid protein first accumulated at the ER and then formed green peroxisomes, but only when intact Pex3 protein was present in the cell.
Then they conducted a test to see if the ER-derived peroxisomes behaved normally. They created a hybrid of thiolase, an enzyme that normally sits inside peroxisomes, and a red fluorescent protein. Without intact Pex3 around, the red thiolase scattered throughout the cell's cytoplasm. In the presence of intact Pex3, however, the newly formed green peroxisomes soon turned yellow, indicating that thiolase had moved in—and that the ER-derived peroxisomes behaved as they ordinarily did. “That was very, very cool,” he says.
More recently, Rachubinski's team reported at the American Society for Cell Biology meeting in December 2007 that shutting off production of two ER proteins in yeast blocks peroxisome formation. “We believe there is special ER machinery” that gives rise to the peroxisome, Rachubinski says.
Photos: Rachubinski: Richard Siemens; Bartel: Donna Carson / AP ©HHMI