George E. Palade: charting the secretory pathway (1999)

For over 50 years, George Emil Palade (Fig. 1) has made fundamental contributions to our understanding of the structure and function of eukaryotic cells, opening new vistas that thousands of scientists continue to explore. He is best known for the integrated morphological and biochemical studies that established the role of the cellular endomembrane system, in particular the endoplasmic reticulum (ER) and the Golgi apparatus, in the synthesis and transport of secretory proteins through the cell1Palade G.E. J. Am. Med. Assoc. 1966; 198: 815-825Crossref Scopus (46) Google Scholar, 2Palade G.E. Science. 1975; 189: 347-358Crossref PubMed Scopus (2507) Google Scholar. This work was recognized with the Nobel Prize, awarded to Palade, together with Albert Claude and Christian de Duve, in 1974. However, Palade’s many other contributions would suffice to secure him a prominent place in the pantheon of modern cell biology. These include his pioneering work as an electron microscopist, which led to many momentous revelations, such as the elucidation of the internal structure of mitochondria, the discovery of ribosomes and the rough ER, the description of the junctional complexes that connect various epithelial cells, and his demonstration of the role of a transcytotic mechanism in transport across the capillary endothelium. As it is impossible to do justice here to all his innovative studies, which continue to this day, I will consider only some of the major discoveries that came from his laboratory when he was at the Rockefeller University. What distinguished Palade from other scientists in the early group of electron microscopists, who, beginning in the mid 1940s, set out to explore and chart the eukaryotic cytoplasm, was his very early awareness that, despite all the exquisite detail it provides, the electron microscope only reveals snapshots of dynamic processes. This led him to undertake a novel combination of electron microscopy, cell fractionation, biochemical analysis, pulse–chase procedures and autoradiography that made it possible for him to elucidate the pathway followed by secretory proteins from their site of synthesis to their site of release at the cell surface. This work inaugurated the field of protein trafficking. Palade was born on 19 November 1912, two years before the beginning of World War I, in Jassy, a Romanian city in the province of Moldavia. He benefited from an early classical education as his father was a professor of philosophy and his mother a teacher. In 1940, he received an MD degree from the University of Bucharest, which included a dissertation on the uriniferous tubules of the porpoise kidney, involving a three-dimensional reconstruction of a nephron from serial sections. In 1946, Palade moved to the USA to pursue a research career in New York city. For a short period, he worked at NYU with Robert Chambers, who was well known for his development of microsurgical techniques to study cellular structure. Chambers, however, retired a few months after Palade’s arrival. Fortunately, Albert Claude gave a seminar at NYU in which he showed some of his first electron micrographs. It was then that he met Palade and invited him to join his group at the Rockefeller Institute (now the Rockefeller University), where Palade was to remain for 28 years. When Palade arrived, Claude had already pioneered the development of cell fractionation, a technique that yielded relatively homogeneous fractions of tissue homogenates in amounts sufficient for biochemical and morphological analysis. A second extraordinary advance from Claude’s laboratory, with the important participation of a brilliant member of the lab, Keith Porter, was the introduction of the electron microscope as a tool to study subcellular structure. In 1945, Porter, together with Claude and Ernest Fullam, published the first electron micrographs of cultured cells, whose periphery was thin enough to be traversed by electrons. The ER had been discovered then, and exploration of the new world that lay below the resolution of the light microscope had begun. During his association with Claude, which ended in 1949 when Claude returned to his native Belgium, Palade was engaged in a cell-fractionation project in collaboration with two biochemists, George Schneider and Walter Hogeboom. The procedure developed by Claude yielded an easily sedimentable ‘large granule fraction’ and a ‘small granule fraction’, later termed ‘microsomes’, whose sedimentation required much higher centrifugal forces. Hogeboom, Schneider and Palade showed that the large-granule subcellular fraction isolated from liver consisted of particles with the properties of mitochondria3Hogeboom G.H. Schneider W.C. Palade G.E. J. Biol. Chem. 1948; 172: 619-637Abstract Full Text PDF PubMed Google Scholar. In this paper, one of his first, Palade introduced the use of sucrose to preserve the morphology of the organelles. His method has since then been almost universally adopted for the preparation of tissue homogenates. At Rockefeller, Claude’s group was part of the laboratory of Pathology and Microbiology, and soon after Claude’s departure, the death of the laboratory director, James B. Murphy, left Porter and Palade in what could have been a precarious situation had the Rockefeller Institute Director himself, Herbert Gasser, not decided to preserve the group under his own direction. Gasser equipped the group with state-of-the-art electron microscopes and supported their work. He asked from his young colleagues little more than their help in preparing and observing his specimens. It was then that George Palade first immersed himself fully in the world of electron microscopy. His first contribution in this field was an improvement of the fixation technique4Palade G.E. J. Exp. Med. 1952; 95: 285-307Crossref PubMed Scopus (871) Google Scholar. It was already clear that OsO4 preserved fine structural details of cultured cells quite adequately, but it was less effective for preserving the structure of cells within tissue blocks. Palade attributed this to the deleterious effects of the uncontrolled acidity of OsO4 solutions and introduced the use of a buffered (veronal acetate) OsO4 fixative. ‘Palade’s fixative’, as it was known, coupled to other technical advances – in particular the introduction of glass knives and plastic embedding materials (methacrylate), and a newly designed microtome by Porter and Blum – resulted in a quantum leap in the quality of electron micrographs obtained after the early 1950s. Palade exploited the technical advances to unveil new details of cellular organization. In 1952, he described in mitochondria the ‘cristae mitochondriales’ as internal ridges or shelves (lamellae) that extend into the mitochondrial matrix5Palade G.E. Anat. Rec. 1952; 114: 427-451Crossref PubMed Scopus (191) Google Scholar. He correctly interpreted them as resulting from infoldings of the inner mitochondrial membrane6Palade G.E. J. Histochem. Cytochem. 1953; 1: 188-211Crossref PubMed Scopus (225) Google Scholar. At about the same time, he discovered the presence in the ground substance of the cytoplasm of a ‘small particulate component’ that came to be known as ‘Palade granules’ and later were named ribosomes. By then, he was collaborating fruitfully with Keith Porter, and in 1954 they jointly described the presence of an ER in cells within tissues7Palade G.E. Porter K.R. J. Exp. Med. 1954; 100: 641-656Crossref PubMed Scopus (113) Google Scholar, similar to that first identified in cultured fibroblasts by Porter and his associates in 1945. In 1955, Palade published an extensive study of the ‘small particulate component of the cytoplasm’ (the ribosomes) that he had identified two years before and correctly forecasted their physiological importance8Palade G.E. J. Biophys. Biochem. Cytol. 1955; 1: 59-68Crossref PubMed Scopus (248) Google Scholar. In this paper, he described the frequent attachment of the particles to the membranes of the ER, which, on account of those particles, became known as ‘rough surfaced membranes’. He also noted that the attached particles were frequently ‘arranged in loops, spirals, circles and rosettes’, which we now know represent polysomes. Palade emphasized that the attached particles were particularly abundant in regions of the cytoplasm that light-microscopists had found to be intensely basophilic and raised the possibility that they contained RNA. In 1955, Palade began a long period of fruitful collaboration with Phillip Siekevitz, whom he recruited to the Rockefeller Institute. With his new associate, Palade decided to undertake the biochemical characterization of the ‘microsomes’, as well as to accomplish the isolation of the ‘small particulate component of the cytoplasm’ that he had described in both its free and membrane-bound forms. Through cell fractionation, biochemistry and electron microscopy, progress was impressive. They soon established that ‘microsomes’ correspond primarily to fragments of the rough ER, which retained the attached particles and were generated in a process of ‘pinching off’ that did not allow for leakage of the cisternal content9Palade G.E. Siekevitz P. J. Biophys. Biochem. Cytol. 1956; 2: 171-200Crossref PubMed Google Scholar. By using a detergent to solubilize the microsomal membranes, they and others were able to recover the attached particles, which were shown to contain protein and RNA and, therefore, were named RNPs (for ribonucleoprotein particles). In the following years, Palade and Siekevitz went on to show that the attached RNPs were the exclusive source of newly synthesized secretory proteins. Although the initial studies on microsomes were carried out with liver, Palade and Siekevitz chose the guinea pig pancreas for their classical work on the secretory pathway. Palade had been impressed by the abundance of the rough ER in the exocrine cells of this organ, which manufactures prodigious amounts of digestive enzymes. His laboratory soon showed that the zymogen granules of the pancreatic exocrine cells serve as temporary storage sites for digestive enzymes10Greene L.J. Hirs C.H.W. Palade G.E. J. Biol. Chem. 1963; 238: 2054-2070Abstract Full Text PDF PubMed Google Scholar. Siekevitz and Palade11Siekevitz P. Palade G.E. J. Biophys. Biochem. Cytol. 1958; 4: 203-218Crossref PubMed Google Scholar, 12Siekevitz P. Palade G.E. J. Biophys. Biochem. Cytol. 1958; 4: 557-574Crossref PubMed Google Scholar, 13Siekevitz P. Palade G.E. J. Biophys. Biochem. Cytol. 1959; 5: 1-10Crossref PubMed Google Scholar, 14Siekevitz P. Palade G.E. J. Biophys. Biochem. Cytol. 1960; 7: 619-630Crossref PubMed Google Scholar developed fractionation procedures for the new system and went on to follow the kinetics of labelling of newly synthesized proteins in subcellular fractions from animals injected with radioactive amino acids. This allowed them to show that the proteins are synthesized on ribosomes bound to the microsomal membranes, next accumulate in the microsomal lumen, which is equivalent to the cavity of the ER cisternae, and then are transported to the zymogen granules themselves. The ground-breaking demonstration from Palade’s laboratory that the Golgi apparatus was a way station for the delivery of proteins from the ER to forming zymogen granules came about because of two innovations. The first was the application to electron microscopy of autoradiography, and the second was the adaptation of an experimental system in which true pulse–chase conditions unattainable in intact animals could be met. The series of papers by Jamieson and Palade that resulted from this work15Jamieson J.D. Palade G.E. J. Cell Biol. 1967; 34: 577-596Crossref PubMed Scopus (537) Google Scholar, 16Jamieson J.D. Palade G.E. J. Cell Biol. 1967; 34: 597-615Crossref PubMed Scopus (374) Google Scholar, 17Jamieson J.D. Palade G.E. J. Cell Biol. 1968; 39: 580-588Crossref PubMed Scopus (156) Google Scholar, 18Jamieson J.D. Palade G.E. J. Cell Biol. 1968; 39: 589-603Crossref PubMed Scopus (232) Google Scholar, 19Jamieson J.D. Palade G.E. J. Cell Biol. 1971; 48: 503-522Crossref PubMed Scopus (164) Google Scholar, 20Jamieson J.D. Palade G.E. J. Cell Biol. 1971; 50: 135-158Crossref PubMed Scopus (268) Google Scholar, employing first pancreatic tissue slices and later isolated lobules, represents landmark studies that demonstrated that, after leaving the ER, the newly synthesized molecules were concentrated in the region of the Golgi apparatus, where they accumulated in developing secretory granules – the ‘condensing vacuoles’. At the same time, Palade’s laboratory was investigating the early stages of synthesis and accumulation of pancreatic enzymes in rough microsomes in vitro. His group showed that isolated membrane-bound ribosomes were capable of carrying out protein synthesis21Kirsch J.F. Siekevitz P. Palade G.E. J. Biol. Chem. 1960; 235: 1419-1424Abstract Full Text PDF PubMed Google Scholar, that the nascent polypeptide chain is associated with the large ribosomal subunit and that this subunit provides a site for ribosome binding to the membrane, with the polypeptide chain strengthening the interaction22Sabatini D.D. Tashiro Y. Palade G.E. J. Mol. Biol. 1966; 19: 503-524Crossref PubMed Scopus (155) Google Scholar. Soon after, the laboratory showed that secretory proteins synthesized by rough microsomes in vitro become sequestered in the microsomal lumen23Redman C.M. Siekevitz P. Palade G.E. J. Biol. Chem. 1966; 241: 1150-1158Abstract Full Text PDF PubMed Google Scholar, and subsequent studies established the vectorial and cotranslational character of the discharge of the nascent polypeptides into the microsomal lumen, providing a foundation for the formulation of the signal hypothesis. While still characterizing the secretory pathway, Palade and Siekevitz also began seminal studies on the structure, biosynthesis and turnover of various organellar membranes, choosing selected membrane proteins for their analysis24Meldolesi J. Jamieson J.D. Palade G.E. J. Cell Biol. 1971; 49: 150-158Crossref PubMed Scopus (65) Google Scholar, 25Palade G.E. Fleischer S. Fleischer B. Methods in Enzymology. Academic Press, 1983: xxixGoogle Scholar, 26Ehrenreich J.H. et al.J. Cell Biol. 1973; 59: 45-72Crossref PubMed Scopus (305) Google Scholar, 27Chua N.H. et al.J. Cell Biol. 1976; 71: 497-514Crossref PubMed Scopus (52) Google Scholar, 28Castle J.D. Palade G.E. J. Cell Biol. 1978; 76: 323-340Crossref PubMed Scopus (34) Google Scholar, 29Scheele G.A. Palade G.E. Tartakoff A.M. J. Cell Biol. 1978; 78: 110-130Crossref PubMed Scopus (63) Google Scholar, 30Howell K.E. Palade G.E. J. Cell Biol. 1982; 92: 822-832Crossref PubMed Scopus (174) Google Scholar. This work extended our understanding of the function of the secretory pathway and membrane biogenesis, clearing the way for the concept that the secretory pathway plays an essential role in the synthesis, topogenesis and distribution of almost all membrane and lysosomal proteins. It also led to the conclusion that cellular membranes do not form de novo but are generated by growth and expansion of preexisting membranes. Palade’s fascination with the structure and function of cellular membranes was not limited to the secretory pathway, and his contributions have enriched many other related aspects of cell biology. For many years, he has had a keen interest in capillary permeability. His earliest studies in this area31Farquhar M.G. Wissig S.L. Palade G.E. J. Exp. Med. 1961; 113: 47-66Crossref PubMed Scopus (349) Google Scholar, 32Farquhar M.G. Palade G.E. J. Exp. Med. 1961; 114: 699-716Crossref PubMed Scopus (170) Google Scholar, performed with Marilyn Farquhar, focused on the glomerular capillaries of the nephron. In subsequent studies, he described the fenestrated endothelium of capillaries in visceral mucosae33Clementi F. Palade G.E. J. Cell Biol. 1969; 41: 33-58Crossref PubMed Scopus (262) Google Scholar and then focused his interest on illuminating the structural basis for the passage of macromolecules across the wall of continuous capillaries, such as those found in muscle. His laboratory discovered the presence of abundant plasmalemmal vesicles (now called caveolae) in endothelial cells and provided evidence that they function as transcytotic carriers, which could also serve as filtering barriers, thus accounting for the two sets of pores postulated by transport physiologists34Palade G.E. Simionescu N. Simionescu M. Endothelial Cell Biology. Plenum Publishing Corp, 1988: 3-22Google Scholar. Palade’s professional association with Farquhar also resulted in two classic papers on the junctional complexes of various epithelia, which stand as a keystone of modern epithelial cell biology35Farquhar M.G. Palade G.E. J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2222) Google Scholar, 36Farquhar M.G. Palade G.E. J. Cell Biol. 1965; 26: 263-291Crossref PubMed Scopus (594) Google Scholar. Having ensured the permanence of cell biology as a discipline at Rockefeller, Palade was attracted by the opportunity of creating a Section of Cell Biology at the Yale University School of Medicine, where in 1973 he moved together with Marilyn Farquhar, who he married in 1970 after the death of his first wife. Under his leadership, that section flowered to become another world centre in membrane biology, focusing mainly on organelle biogenesis and protein traffic, as he recruited outstanding young researchers – now world leaders in this field. After 17 years at Yale, Palade and Farquhar moved again, this time to accept positions at UCSD, where in the past nine years they have been a major force in establishing a Division of Cellular and Molecular Medicine. At San Diego, Palade has continued his active research on capillary permeability, while maintaining a profound interest, shared with Farquhar, in membrane and protein traffic and, in particular, the function of the Golgi apparatus37Farquhar M.G. Palade G.E. J. Cell Biol. 1981; 91: 77s-103sCrossref PubMed Google Scholar, 38Farquhar M.G. Palade G.E. Trends Cell Biol. 1998; 8: 2-10Abstract Full Text PDF PubMed Scopus (225) Google Scholar (Fig. 2). I could not conclude this summary of Palade’s major contributions without dwelling, albeit briefly, on his personal attributes that make him one of the most admired and beloved figures in today’s scientific scene. Palade not only has a powerful intellect that allows him quickly to cut to the essence of a scientific problem and propose for it a feasible solution but is also a man of great human – and and He is also a with a keen and an extraordinary and of of and classical His and for make some of his scientific of the development of our field in this and the the years, I have had the of with him many the and in of them he was a who could to past and He is a man who in which he has to the of cell biology. His of allowed him to a series of at the de and his with is such that he could out to a and the of – which in we in – and the of which he could by As a and as a Palade has been an source of and which he with a and while still the of I with a we made to where he as an who a to remain in the USA and accept a at The was not only because in the had to its first but also the Palade the time to make a of his with their past and which I was what most impressed the The and of George Palade have and, in the Palade new of cell are the modern technical to from the eukaryotic cell the new that its structure still I that, to I have not the contributions of many of Palade’s however, are of Palade’s in which he their work. I to for his help and in preparing this