Chapter 8. Movement Within The Endomembrane System8.1. Discovery of the secretory pathwayWe have seen that cells are composed of a multitude of membranous motifs, including the ER, the Golgi apparatus, the vacuole and the plasma membrane. In this chapter I will discuss the relationships of the various membrane systems to each other and how they are generated (Claude, 1970; Griffiths, 1996). The relationships between the various membranes were revealed to a large extent by studying the secretory process in pancreatic exocrine cells.Henri Dutrochet (1824, in Schwartz and Bishop, 1958) postulated that “it is within the cell that the secretion of the fluid peculiar to each organ is effected….The cell is the secreting organ par excellence. It secretes, inside itself, substances which are, in some cases, destined to be transported to the outside of the body by way of the excretory ducts, and, in other cases, destined to remain within the cell which has produced them….” In the eighteen seventies and eighties, Rudolf Heidenhain (1878) studied the exocrine cells of the pancreas of mammals. He noticed that shortly after an animal ate, microscopic granules disappeared from the apical part of their pancreatic cells, and reappeared a few hours later. He correlated the disappearance of the apical granules with the appearance of digestive enzymes in the pancreatic juices that he measured biochemically and concluded that the granules, which he dubbed zymogen granules, contained the precursors of the digestive enzymes. The zymogen granules, he supposed, represented an available store of digestive enzymes that could be released upon eating.Limited by technology, Heidenhain was unable to elucidate the intracellular pathways involved in the secretion of the chymotrypsinogen, trypsinogen and  amylase that are stored in the zymogen granules. However,impressed with Heidenhain’s work that combined morphology with biochemistry, George Palade (1959) set out to understand the intracellular partof the secretory process using, and more importantly, integrating the newly developed techniques of electron microscopy and cell fractionation. Palade (1959) considered these studies to be “a collaboration over almost a century 257between Rudolf Heidenhain, Philip Siekevitz, and myself.” The integrated studies by Palade and his colleagues have become a watershed in the study of the intracellular secretory pathway. These papers constitute a good pedagogical example of the interplay between theory and experiment, inductive and deductive reasoning, and technique and interpretation.While all cells secrete one thing or another, Palade chose to study secretion in cells that specialized in secretion. Palade and his collegues injected 3H-leucine into guinea pigs and then rapidly isolated the pancreas. Siekevitz and Palade (1958a,b,c,1959,1960a,b), using subcellular fractionation techniques; and Caro and Palade (1964), using radioautographyat the EM level, extended Heidenhain’s conclusion by showing that the digestive enzymes, stored in the smooth membrane enclosed zymogen granules at the apical end of pancreatic exocrine cells, were synthesized on the rough endoplasmic reticulum at the basal region of the cell (Figures 8-1, 8-2, 8-3). However their ability to resolve the pathway followed by the digestive enzymes as they moved from the rough membranes at the basal portion of the cell to the smooth membrane enclosed vesicles at the apical region of the cell was compromised by the fact that it took too much time to label the newly synthesized proteins by intravenously supplying the pancreaswith radioactive amino acids.The lack of temporal resolution was overcome by Jamieson and Palade (1966,1967a,b,1968a,b,1971a,b) who switched from whole guinea pigs to pancreatic tissue slices. This minimized the time it took for the tracer to travel to, and to diffuse into, the site of incorporation; since the time it takes to diffuse into the exocrine cells is minimized when the surface to volume ratio is maximized. Moreover, thin sections maximized the uniformity of labeling in each cell since the tracer enters all the cells at approximately the same time. Now Jamieson and Palade were able to see a precursor-product relationship between organelles just as biochemists had seen in chemical reactions. Tissue slices had previously been used successfully by many biochemists, including Otto Warburg, Albert Szent-Györgyi and Hans Krebs to maximize the temporal resolution necessary to determine the sequences ina pathway.Jamieson and Palade labeled the cells for only three minutes with radioactive leucine and then replaced the radioactive leucine with an excess concentration of cold leucine. They followed the movement of label with 258both electron microscopic radioautography and by cell fractionation. They discovered that the label moved like a wave through the cell. It started at theribosomes on the ER, which represent the site of protein synthesis. The protein then entered the lumen of the ER, and was only released by treatments that break the membranes. After 7 minutes the label appeared in the peripheral vesicles of the Golgi apparatus. The labeled protein probably took a membranous route from the ER to the Golgi apparatus since the label never increased in the cytosolic fraction. Moreover, the labeled protein traveled from lumen to lumen, since it could only be released from the Golgimembranes by high pH treatments that destroyed membrane integrity. Thirty-seven minutes following the pulse-label, the protein appeared in the condensing vacuoles at the trans-Golgi network. Approximately 2 h after thepulse-label, essentially all the labeled protein was in the zymogen granules atthe apical end of the cell where they are stored. Thus the fact that they selected favorable material for studying secretion, combined with their biophysical insight in deciding to use tissue slices in order to obtain a uniform and rapid uptake of labeled amino acids, allowed Jamieson and Palade to see that the movement of proteins from the ER to the Golgi apparatus.Interestingly, treating the tissue slices with chemicals that stimulate secretion caused an increase in secretory flow by increasing the amount of protein secreted, not by increasing the rate of movement of a given protein through the secretory pathway. Thus there must be an increase in the rate of protein synthesis and/or the area of the pathway. Indeed, the secretion-stimulating agents