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Classic Experiment 17.1 Following a Protein Out of the Cell T he advent of electron microscopy allowed researchers to see the cell and its structures at an unprecedented level of detail. George Palade utilized 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 of the cell. The mechanism of protein secretion fascinated many cell biologists, including Palade. In early research on secretion, cells were disrupted and the various organelles separated by centrifugation. These cell fractionation studies had shown that secreted proteins are present in membranebound vesicles associated with the endoplasmic reticulum (ER), where they are synthesized, and with the plasma membrane, where they are eventually released from the cell. Unfortunately, results from these studies were hard to interpret due to difficulties in obtaining clean separations between different organelles. To further clarify the pathway, Palade turned to a newly developed technique, highresolution autoradiography, that allowed him to follow radioactively labeled proteins as they moved within the cell. 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 which cell structures and organelles participate in protein secretion. To study such a complex process, he carefully chose an appropriate model system for his studies, the pancreatic exocrine cell, which is responsible for producing and secreting large amounts of digestive enzymes. Palade first examined the protein secretion pathway in vivo by injecting 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, 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 the labeled proteins were in cells at various times. As expected, the radioactivity localized in vesicles at the ER at time points 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 never was a time point where 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 a radiolabeled precursor, in this case [3H]leucine, for a short period of time known as the “pulse.” The radioactive precursor is then replaced with its (a) nonlabeled form for a subsequent “chase” period. Proteins synthesized during the pulse period will be labeled and detected by autoradiography, while those synthesized during the chase period, being nonlabeled, will not be detected. Palade began by cutting the guinea pig pancreas into thick slices, which were then incubated for 3 minutes in media 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 assure 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, he pro(b) (c) (d) Figure 17.1 The synthesis and movement of guinea-pig pancreatic secretory proteins as revealed by electron microscope autoradiography. (a) At the end of a 3-minute labeling period with [3H]leucine, the tissue is fixed, sectioned for electron microscopy, and subjected to autoradiography. Most of the labeled proteins (the autoradiographic grains) are over the rough ER. (b) Following a 7-minute chase period with unlabeled leucine, most of the labeled proteins have moved to the Golgi vesicles. (c) After a 37-minute chase, most of the proteins are over immature secretory vesicles. (d) After a 117-minute chase, the majority of the proteins are over mature zymogen granules. [Courtesy of J. Jamieson and G. Palade.] ceeded 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 in 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 Figure17.1). 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 proteins traveling through the secretory pathway. His studies on the pancreatic exocrine cells yielded two semi- nal observations. First, 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, secreted proteins never mix with other cellular proteins; they are segregated into vesicles throughout the 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 the choice of a cell type devoted to secretion, pancreatic exocrine cell, as a model system. In a different cell type, significant amounts of nonsecreted proteins might have also been produced during the pulse, possibly leading to ambiguous results. Palade’s work set the stage for more detailed studies. Once the secretory pathway was clearly described, entire fields of research were opened up to investigation, in the synthesis and movement of both secreted and membrane proteins. For this ground-breaking work, Palade was awarded the Nobel Prize for Physiology and Medicine in 1974.