Following a Protein Out of the Cell

J. Jamieson and G. Palade, 1966, P. Natl. Acad. Sci. USA 55(2):424–431

The advent of electron microscopy allowed researchers to see the cell and its structures at an unprecedented level of detail. George Palade used this tool not only to look at the fine details of the cell, but also to analyze the process of secretion. By combining electron microscopy with pulse-chase experiments, Palade uncovered the path proteins follow to leave the cell.

Background

In addition to synthesizing proteins to carry out cellular functions, many cells must also produce and secrete additional proteins that perform their duties outside the cell. Cell biologists, including Palade, wondered how secreted proteins make their passage from the inside to the outside of the cell. Early experiments whose results suggested that proteins destined for secretion are synthesized in a particular intracellular location and then follow a pathway to the cell surface employed methods to disrupt cells synthesizing a particular secreted protein and to separate their various organelles by centrifugation. These cell-fractionation studies showed that secreted proteins can be found in membrane-bounded vesicles derived from the endoplasmic reticulum (ER), where they are synthesized, and within zymogen granules (regulated secretory vesicles), from which they are eventually released from the cell. Unfortunately, results from these studies were hard to interpret due to difficulties in obtaining clean separation of all the different organelles that contain secretory proteins. To further clarify the secretory pathway, Palade turned to a newly developed technique, high-resolution autoradiography, that allowed him to detect the positions of radioactively labeled proteins in thin cell sections that had been prepared for electron microscopy of intracellular organelles. His work led to the seminal finding that secreted proteins travel within vesicles from the ER to the Golgi complex and then to the plasma membrane.

The Experiment

Palade wanted to identify the cell structures and organelles that participate in protein secretion. To study this complex process, he carefully chose an appropriate model system, the pancreatic acinar cell, which is responsible for producing and secreting large amounts of digestive enzymes. Because these cells have the unusual property of expressing only secretory proteins, a general label for newly synthesized protein, such as radioactively labeled leucine, would only be incorporated into protein molecules that were following the secretory pathway.

Palade first examined the protein secretory pathway in vivo by injecting live guinea pigs with [³H]leucine, which was incorporated into newly made proteins, thereby radioactively labeling them. At time points from 4 minutes to 15 hours after injection, the animals were sacrificed and the pancreatic tissue was fixed. By subjecting the specimens to autoradiography and viewing them in an electron microscope, Palade could trace where in cells the labeled proteins were located at various times. As expected, the radioactivity was localized in vesicles at the ER immediately following the [³H]leucine injection and at the plasma membrane at the later time points. The surprise came in the middle time points. Rather than traveling straight from the ER to the plasma membrane, the radioactively labeled proteins appeared to stop off at the Golgi complex in the middle of their journey. In addition, there was no time point at which the radioactively labeled proteins were not confined to vesicles.

The observation that the Golgi complex was involved in protein secretion was both surprising and intriguing. To thoroughly address the role of this organelle in protein secretion, Palade turned to in vitro pulse-chase experiments, which permitted more precise monitoring of the fate of labeled proteins. In this labeling technique, cells are exposed to radiolabeled precursor, in this case [³H]leucine, for a short period known as the pulse. The radioactive precursor is then replaced with its unlabeled form for a subsequent chase period. Proteins synthesized during the pulse period will be labeled and detected by autoradiography, whereas those synthesized during the chase period, which are not labeled, will not be detected. Palade began by cutting guinea pig pancreas into thick slices, which were then incubated for 3 minutes in a medium containing [³H]leucine. At the end of the pulse, he added excess unlabeled leucine. The tissue slices were then either fixed for autoradiography or used for cell fractionation. To ensure that his results were an accurate reflection of protein secretion in vivo, Palade meticulously characterized the system.

Once convinced that his in vitro system accurately mimicked protein secretion in vivo, Palade proceeded to the critical experiment. He pulse-labeled tissue slices with [³H]leucine for 3 minutes, then chased the label for 7, 17, 37, 57, and 117 minutes with unlabeled leucine. Radioactivity, again confined to vesicles, began at the ER, then traveled in vesicles to the Golgi complex and remained in the vesicles as they passed through the Golgi and onto the plasma membrane (see Figure 1). As the vesicles traveled farther along the pathway, they became more densely packed with radioactive protein. From his remarkable series of autoradiograms at different chase times, Palade concluded that secreted proteins travel in vesicles from the ER to the Golgi and onto the plasma membrane, and that throughout this process they remain in vesicles and do not mix with the rest of the cell.

Discussion

Palade’s experiments gave biologists the first clear look at the stages of the secretory pathway. His studies on pancreatic acinar cells yielded two fundamental observations. First, he showed that secreted proteins pass through the Golgi complex on their way out of the cell. This was the first function assigned to the Golgi complex. Second, he showed that secreted proteins never mix with cellular proteins in the cytosol; they are segregated in vesicles throughout the secretory pathway. These findings were predicated on two important aspects of the experimental design. Palade’s careful use of electron microscopy and autoradiography allowed him to look at the fine details of the pathway. Of equal importance was his choice of a cell type devoted to secretion, the pancreatic acinar cell, as a model system. In a different cell type, significant amounts of nonsecretory proteins would have also been produced during the labeling, obscuring the fate of secretory proteins in particular.

Palade’s work set the stage for more detailed studies. Once the secretory pathway had been clearly described, entire fields of research were opened up to investigation of the synthesis and movement of both secreted and membrane proteins. For this groundbreaking work, Palade was awarded the Nobel Prize in Physiology or Medicine in 1974.