Choose the right experimental system to test a hypothesis: that has and always will be one of the ten commandments of cell biology. By the mid-1960s, several secretory cell types—liver, pancreatic, pituitary—had been exploited for tracking the path and fate of their abundant loads of secreted proteins. And by 1964, these cells had revealed a sketch of the secretory pathway from the ER to the Golgi, to vesicles, and out to the cellular membrane. This was most clearly seen when Caro and Palade (1964) combined autoradiography and electron microscopy to trace radioactive leucine in pancreatic cells.
After injecting the rats with tritiated glucose, Neutra and Leblond (1966a) saw the radioactive sugars concentrating exclusively in the Golgi at early time points and then moving out to the mucigen granules later. The results were confirmed with other sugars and other cell types (Neutra and Leblond, 1966b). “The results were striking,” says Neutra, and led to the conclusion that the Golgi is “the site where simple sugars become immobilized on larger molecules.”
Neutra says the discovery that glycoproteins that remain associated with the cell surface are also glycosylated in the Golgi was an “unexpected” bit of luck arising from the study of goblet cells in the intestinal epithelium. In the micrographs, she saw that the columnar cells next to the goblet cells were also concentrating the radioactive sugars in the Golgi. At later time points the columnar cells showed a border of glycoprotein on their apical cell surfaces. “By examining a wide variety of tissues and organs, we were able to show for the first time that glycosylation probably occurs in the Golgi in all cells,” she says.
The results nicely matched the recent findings that the Golgi was the site of another protein modification, sulfation (Lane et al., 1964; Godman and Lane, 1964). Follow-up work included the demonstration of a 40-fold enrichment of galactosyl transferase activity in isolated Golgi membranes, confirming that glycosylating enzymes were in the right place to do the job (Fleischer et al., 1969). As additional radioactive sugars such as mannose and fucose became available, Leblond's laboratory showed that there was a division of labor between cellular compartments. Mannose, the group found, is added to thyroglobulin in the ER, and then galactose and fucose are added subsequently in the Golgi complex (Whur et al., 1969; Haddad et al., 1971).
Neutra says her graduate studies of the intestinal epithelium led her to a research career focused on mucosal immunology and microbial–epithelial interactions in the intestine. But she cherishes being a part of “an extremely fun time in cell biology”—a time that was fun, “partly because of the technical limitations,” she says. “You couldn't design recombinant molecules. The wonderful thing at that time was the beauty of the intracellular structures still being discovered.”