Change Is Good: Life Outside the Nucleus

TURNING points

Change is good: life outside the nucleus

Randy Schekman

No one knows what the future holds, but one great fortune of becoming Kornberg’s gradu- in the structure of native membranes led S. thing is clear, everything changes. The best ate student; thus making the first important J. Singer and Nicholson to propose a fluid advice for a young scholar is to remain flex- change in my career — from using a primarily mosaic model with lipids and membrane ible and seek new experiences, unbound by in vivo to an in vitro approach to study a com- proteins free to diffuse laterally within the past successes or failures. The temptation is to plex cellular process. plane of the bilayer. stick with what works, but to be independent In Kornberg’s laboratory, I teamed up with In another corner of the cell biology in the research world, change is good. Doug Brutlag and Bill Wickner to develop a world, regulation of cell division yielded to I was ignorant of this view when I began method to define the physiologically rele- an elegant genetic dissection developed by my research career as an undergraduate vant dna gene products essential for conver- Lee Hartwell. If such a classic approach could student at the University of California, Los sion of ϕX174 single stranded DNA to the illuminate the genes and proteins that govern Angeles. Fate brought me in touch with a replicative form. We succeeded in produc- progress through the cell cycle, then surely a new assistant professor, Dan Ray of the then ing a soluble E. coli lysate that was compe- similar effort could help open the secretory Zoology Department. During this period, tent for replication, but which was defective pathway to functional analysis. And combin- I developed an interest in the replicative when replication mutant cells were used as a ing classical genetics with biochemical recon- mechanism of M13 and ϕX174 phage DNA, source of cytosol. This observation permit- stitution could perhaps lead to mechanistic a field that was essentially created by the pio- ted us to isolate functional forms of several insights in an area as complicated as mem- neering biophysicist Robert Sinsheimer at of the replication proteins, including an brane assembly, as it had in DNA replication the California Institute of Technology. The RNA primase and the DNA polymerase III and bacteriophage particle assembly even burning question at the time was how a sin- holoenzyme, and it led Kornberg and, in a earlier. The opportunities presented by yeast gle-stranded circular DNA template could parallel effort, Jerard Hurwitz to claim suc- as a model eukaryote and the nascent area of be copied to a double-stranded replicative cess several years later by completely recon- membrane assembly were sufficiently alluring form, and then how this replicative form stituting replication with pure proteins and to encourage me to make a clean break from could spool-out new single-stranded circular a DNA template. nucleic acids. progeny. The analytic tool was genetics along- I was powerfully impressed by this success- What better time to make a change than side density and velocity gradient separation ful model of how to approach a problem, but after an intense period of training under the of replicative intermediates. put off by the fierce competition in the field influence of a powerful mentor. I jumped At the same time, using a classic enzymologi- of DNA replication at the time. Although my ship to mammalian membranes during cal approach, Arthur Kornberg and his post- training and inclination had prepared me for a two-year postdoctoral stint with S. J. doctoral fellow Mehran Goulian had succeeded a career in nucleic acid research, I was drawn Singer, and then again to yeast genetics and in converting ϕX174 single-stranded circles to by the allure of an emerging area of research in membrane biochemistry when I moved to a double-stranded form using Escherichia coli cellular membranes. Berkeley to begin my independent career in DNA polymerase (now called polymerase I) In the early 1970s, membrane cell biol- 1976. Of course, such change is not without and a crude source of DNA oligonucleotide ogy remained in a descriptive phase. George risk; my first NIH grant proposal to work primers obtained by heating an E. coli lysate. Palade and his colleagues had brilliantly on yeast membranes was trashed because I I was convinced that this sort of biochemi- charted the path of secretion in pancreatic had no relevant experience or preliminary cal approach would eventually clarify the exocrine cells, yet mechanistic aspects of data. But with great students and colleagues mechanism of DNA replication. I had the this process remained shrouded. At the same at Berkeley, my laboratory and those of sev- time, the first biochemical approaches to eral former students succeeded in defining Randy Schekman is Professor of Cell and synthesize secretory proteins and reconsti- the secretory pathway with genetics and then Developmental Biology in the Department of tute protein translocation in a cell-free reac- by reconstituting and purifying many com- Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA. tion emerged in the laboratories of Blobel, ponents required for vesicular traffic. I have e-mail: [email protected] Milstein and Sabatini. Other developments never regretted the change.