Philip Siekevitz: Bridging biochemistry and cell biology.

Philip Siekevitz, an Emeritus Professor at the Rockefeller University who made pioneering contributions to the development of modern cell biology, passed away on December 5th, 2009. He was a creative and enthusiastic scientist, as well as a great experimentalist who throughout his lifetime transmitted the joy of practicing science and the happiness that comes with the acquisition of new knowledge. He was a man of great integrity, with a thoroughly engaging personality and a humility not often found in people of his talent.

Philip Siekevitz’s career proceeded along three phases marked byseminal contributions that opened up new avenues of research. The first phase was in thefield of protein synthesis, in which he developed the first in vitro system usingdefined cell fractions. Then, in collaboration with George Palade, he demonstrated thecentral role of ribosomes in protein synthesis and, in particular, of membrane-boundribosomes in the synthesis of secretory proteins. In later stages of his careerSiekevitz turned his attention to the nervous system, where he isolated andcharacterized the postsynaptic density, a structure that integrates the activity of manytransmembrane and associated proteins in mediating synaptic transmission.

Philip Siekevitz

IMAGE C/O THE ROCKEFELLER UNIVERSITY

Early years

Siekevitz was born in 1918 in South Philadelphia, where his immigrant father was askilled worker in a garment factory and his mother was a dressmaker. His interest inbiology began in high school, but he was a child of the Depression and upongraduation spent two years working to earn the funds necessary to pay for a collegeeducation. He attended the Philadelphia College of Pharmacy and Science, where hedeveloped an interest in biochemistry. However, soon after graduation, in 1942, heentered the Army and served in a decontamination unit prepared to respond tochemical warfare attacks. Because he was eager to enhance his scientific background,Siekevitz requested a transfer, which resulted in his deployment as a laboratorytechnician to an Air Force Supply Base for the Pacific War in San Bernardino,California, where he honed his skills in microscopy and chemical analysis.

In 1945, after three and a half years in the service, Siekevitz was admitted to thegraduate program of the already highly regarded Biochemistry Department of theUniversity of California, then at Berkeley, where his tuition and living expenseswere provided by the GI Bill.

David Greenberg, head of the program and Phil’s PhD thesis advisor,recalled later that the department had gradually contracted during the war yearsbut, with peace, turmoil descended upon it in the form of a huge influx of beginnersall of whom, like Phil, had hiatuses in their careers. Phil was attracted to themysteries of protein synthesis, but Greenberg steered him to the study of themetabolism of the amino acids glycine and serine in liver slices, for whichSiekevitz took advantage of the availability of 14C produced in a nearbycyclotron.

Protein synthesis in vitro

In 1949 Phil turned 31, received his PhD, and married Rebecca Burstein, who remainedhis wife for 60 years. The Siekevitzes then moved to Boston, where Phil, with apublic health service fellowship, joined the group of Paul Zamecnik at theHuntington Laboratories of Harvard University at the Massachusetts General Hospital.Zamecnik was a major contributor to the elucidation of the molecular events inprotein synthesis. Siekevitz threw himself with enthusiasm into this field at a timein which only three laboratories in the country were beginning to explore itsbiochemical mysteries.

Up to then, most work on protein synthesis had been performed by measuring theincorporation of radioactive amino acids into proteins in whole animals or in tissueslices incubated in vitro because acellular homogenates had shown minimal activity.Siekevitz achieved a breakthrough by taking advantage of cell fractionationtechniques. In later work at the Rockefeller Institute, he developed this techniqueto new heights and called it “a bridge between the morphologists and thebiochemists.” In a collaboration with Zamecnik, and in an impressive andcomprehensive paper (Siekevitz, 1952) ofwhich he was the sole author, Siekevitz first related biochemical requirements torecognizable structures. He examined the protein-synthesizing activity of an invitro system in which he combined in various ratios mitochondrial, microsomal, andsupernatant-derived fractions. Using this approach, he demonstrated the synergisticrole of energy-producing mitochondria in sustaining the activity of microsomes,which showed the highest rate of incorporation of labeled amino acids into theirproteins. In Siekevitz’s system mitochondria were fueled byα-ketoglutarate or succinate, which sustained the production by themitochondria of a soluble factor that enabled the microsomes to incorporate alanineinto its proteins. Because Siekevitz showed that the factor was consumed whenhexokinase was added to the system, he suggested that the factor could be ATP, theuniversal energy currency discovered by Fritz Lippman.

Phil’s system was the forerunner of many other advances inZamecnik’s laboratory, and in other laboratories, which elucidated themechanism of amino acid activation (Hoagland etal., 1956). Two years after Siekevitz’s paper, Zamecnik andElizabeth Keller (Zamecnik and Keller,1954) introduced a major simplification by replacing the mitochondria with asoluble ATP-generating system. Further biochemical dissection inZamecnik’s laboratory of the supernatant-derived fraction led to themomentous discovery of tRNA (Hoagland et al.,1958).

Siekevitz’s discovery of the role of the mitochondrion as the energysupplier for protein synthesis led him to apply for an oncology fellowship to workwith Van Potter, then a pioneer of research on energy metabolism, at the McArdleCancer Laboratories of the University of Wisconsin in Madison, the campus wherePhil’s wife, Rebecca, had been a student. It was during this fellowshipthat their two daughters, Ruth and Miriam, were born.

Siekevitz’s time with Van Potter (1951–1954) was highlyproductive, with him publishing six papers on the enzymology andcompartmentalization of adenine nucleotide metabolism, all but one of them inThe Journal of Biological Chemistry.

The central biosynthetic role of the ER

Keith Porter and George Palade, who at the Rockefeller Institute were makingtrailblazing discoveries with the electron microscope on the identification andstructure of subcellular organelles, were among the many who appreciatedSiekevitz’s development of an in vitro protein-synthesizing system. Infact, Porter already cited Siekevitz in a 1953 paper in The Journal ofExperimental Medicine (Porter,1953), and in 1954, Palade invited Siekevitz to present his work at theRockefeller Institute. During this visit Siekevitz was easily persuaded to joinPalade’s group.

Together, Siekevitz and Palade undertook what has been referred to as “thechemical dissection of the microsomes,” as well as the elucidation of thefunction of the small particles that Palade had observed in the electron microscopeeither free in the cytoplasm or bound to the cisternal membranes of the roughportions of the endoplasmic reticulum (ER; Palade,1955). In a masterful combination of cell fractionation, biochemistry,and electron microscopy (Palade and Siekevitz,1956a,b), the team establishedthat microsomes arise by a peculiar fragmentation of the ER during which membranevesicles with their attached particles pinch off from ER cisterna without leakage ofthe cisternal content (Fig. 1). In a study ofliver mitochondria that Siekevitz was carrying out with Michael Watson, he had usedsodium deoxycholate to solubilize mitochondrial membranes and found that thisdetergent also effectively solubilized microsomal membranes—allowing forthe recovery of the attached particles by high-speed centrifugation. The particleswere shown to be rich in protein and RNA, and hence, were named RNPs forribonucleoprotein particles.

Figure 1.

Microsomes are fragments of the rough ER. (Left) Electronmicrograph showing the cytoplasm in an acinar cell of the guinea pigpancreas. rs, rough ER; ss, smooth ER; m, mitochondria. (Right) An EMsection of a microsome pellet isolated from pancreatic cells. Note thepresence of various kinds of membrane-bound vesicles (mc1,mc2, and mc3), most of which are studded withdense particles that were later identified to be ribosomes. dm, damagedmitochondria; g, dense granules. From Palade and Siekevitz (1956b).

Although the initial work on microsomes was performed in the liver, Palade andSiekevitz chose the guinea pig pancreas for their subsequent work because Palade hadrecognized the extraordinary development of the ER in the acinar cells of thisorgan, which manufactures prodigious amounts of digestive enzymes. It was with thissystem that in subsequent seminal papers Siekevitz and Palade showed that themembrane-bound RNPs, later named ribosomes, were the exclusive site of synthesis ofpancreatic enzymes (Siekevitz and Palade,1958b,c).

Palade’s and Siekevitz’s further studies focused on thekinetics of labeling of secretory proteins recovered in different subcellularfractions after labeling in vivo with injected radioactive amino acids (Siekevitz and Palade, 1958a, 1959, 1960). This allowed them to show that pancreatic enzymes synthesized inmembrane-bound ribosomes—in vivo, as well as in vitro with incubatedmicrosomes (Redman et al.,1966)—subsequently accumulate in the lumen of the microsomalvesicles from where they could be released after membrane solubilization. This worklaid the foundation for the subsequent studies by Jim Jamieson and Palade thattraced the pathway of newly synthesized pancreatic enzyme precursors from the ER tothe Golgi apparatus, where they are concentrated in zymogen granules to bedischarged at the cell surface.

At Rockefeller, Siekevitz developed a strong interest in membrane biochemistry and,in particular, in organellar membrane biogenesis. In following years he and theyoung scientists who joined his laboratory played a major role in studies of thebiosynthesis, structure, and function of the ER, chloroplast, and neuronal membraneproteins.

Siekevitz’s work on the postsynaptic density (PSD), which began in themid-1970s—like his earlier research on the protein-synthesizing apparatusof secretory cells—was driven by his ability to devise approaches toisolate subcellular structures first identified in situ by electron microscopy andto characterize them biochemically. The postsynaptic density had been visualized inthe 1950s, and Siekevitz and co-workers refined existing procedures for theirisolation by detergent treatment of synaptosomal fractions. This allowed his groupto identify within the PSD signaling molecules and ion channels involved in nerveimpulse transmission (for example, see Wu et al.,1985, 1992). It is nowrecognized that the PSD represents a macromolecular assembly that organizes thepostsynaptic signaling machinery. In a theoretical paper (Siekevitz, 1985), Siekevitz presciently postulated thatlong-lasting changes in neuronal circuitry result from “changes in theconcentration and conformation of PSD proteins, changes that could alter theneurophysiology of dendritic spines.”

A man with a roving intellect and deep ethical convictions

Siekevitz had many admirable personal attributes that endeared him to those in thebiochemistry and cell biology communities with whom he interacted and, inparticular, to those, like myself, whom he hosted in his laboratory and verygenerously advised and supported without expecting recognition of his influence orhis contributions.

Siekevitz was also a highly principled person who adhered to the highest standards ofprofessional and personal behavior. His publications are shining examples of how hewent out of his way to give due credit to others who might have preceded him withdiscoveries in the field of his work.

Siekevitz’s concern for ethical issues impinging on the behavior ofscientists led him to often write articles and letters to the Editor in journals andnewspapers, ranging from Nature, Science,The Scientist, and The Nation to TheNew York Times. He commented on issues related to science and society,including the hubris of scientists who neglected their responsibility to inform thepublic of the implications of their research. He was particularly concerned that inthe era of rapid biotechnological advances, scientists were tempted to profitinordinately from discoveries that were made with public funds. He regretted that“the mixture of science and money” was poisoning theatmosphere of free inquiry and disinterested cooperation in which science thrivesbest. He also feared that competition for important scientific prizes was fosteringsecrecy and preventing due recognition of the research achievements made by othersworking in the same area. These grave concerns did not diminishSiekevitz’s amiable collegiality or his willingness to generously givehis time to others and to share his knowledge, as much with important peers as withyounger beginners who sought his advice or counsel.

Siekevitz had a fertile mind and an insatiable and roving intellectual curiosity. Hedid not limit his intellectual pursuits to the biological sciences, but read avidlyin the physical and social sciences. Beyond the over 120 papers that reported hisscientific contributions, he found time to write with Ariel Loewy in 1963,“Cell Structure and Function,” the first American textbook incell biology, which underwent two more editions. He had literary talents andpublished in New Directions Press two fictional novellas, “ThePetition” in 1948, and “The Fish” in 1950. Heplayed the piano, with Mozart and Beethoven being his favorites, and in 1988 beganto write a series of fictional short stories on Mozart, his family, his operas, andtheir characters, all of them yet unpublished.

For his scientific achievements, Siekevitz gathered too many honors to be listedhere. He was President of the American Society for Cell Biology in 1966, beingpreceded by Van Potter and George Palade, and of the New York Academy of Sciences.He was elected to the National Academy of Sciences in 1975 and chaired the sectionon cellular and developmental biology. He was a fellow of the American Academy ofArts and Sciences and of the American Association for the Advancement of Science. Hereceived honorary degrees from his college Alma Mater and from the University ofStockholm in 1974. He was the editor of the JCB from 1961 to 1964and served in editorial boards of many other journals.