Charting the secretory pathway in a simple eukaryote.

George Palade, a founding father of cell biology and of the American Society for Cell Biology (ASCB), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. His vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. My laboratory has dissected these pathways with complementary genetic and biochemical approaches. Peter Novick, one of my first graduate students, isolated secretion mutants of Saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding SEC genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. A biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize Sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (ER) membrane (Sec61) and the cytoplasmic coat protein complex (COPII) that captures cargo proteins into transport vesicles that bud from the ER.

GEORGE PALADE FRAMES THE QUESTIONS

The myriad internal membrane structures that characterize eukaryotic cells were well known to nineteenth century cytologists, notably Camillo Golgi (Golgi, 1898). However, with the perfection of techniques to preserve membranes for ultrastructural analysis by transmission electron microscopy, George Palade focused our attention on the role of intracellular membranes in the maturation and secretion of proteins manufactured by cells of the exocrine pancreas. His pulse-chase autoradiographic tracing of newly synthesized zymogen proteins, marching progressively from the endoplasmic reticulum (ER) through the Golgi complex and storage granules to the cell surface, charted a pathway that only hinted at the complexity of intracellular membrane transactions (Palade, 1975). Nonetheless, in spite of the limitations of the technology, Palade framed the basic questions that have engaged a generation of membrane biologists, biochemists, and geneticists. Before any precise molecular probes became available, Palade recognized that membrane assembly was achieved by expansion of a preexisting organelle, as opposed to the de novo creation of a membrane, and that traffic was mediated by transport carriers, vesicles that capture secretory cargo en route to the next station in the pathway.

Randy Schekman

EARLY INFLUENCES AND THE DEVELOPMENT OF A GENETIC APPROACH TO SECRETION IN YEAST

I learned of Palade's work in graduate school, but I was not trained as a cell biologist. My background was in the enzymology and physiology of bacterial DNA replication. My mentor Arthur Kornberg greatly admired Palade's work, although the two had completely different styles and approaches. In considering where to apply my training in biochemistry, I was influenced by S. Jonathan Singer's ideas about the dynamic structure of cellular membranes and Lee Hartwell's genetic dissection of the yeast division cycle. After postdoctoral training in Singer's lab, I decided to focus my independent work on protein secretion in yeast.

After my first year at UC Berkeley, Peter Novick joined the lab as a beginning graduate student. He and I embarked on a search for conditional lethal, temperature-sensitive (ts) secretion mutants. After an aborted attempt to apply a “clever” genetic selection (Schekman and Novick, 2004), Peter decided to inspect a random assortment of ts isolates, one of which, called sec1, proved to be defective in the last step of secretion as seen by the intracellular accumulation of mature secretory proteins and vesicles (Novick and Schekman, 1979). Peter then found that sec1 mutant cells experience a dramatic increase in buoyant density that allowed him to use density gradient separation to enrich for more mutants that accumulate ER, Golgi structures, or vesicles at a restrictive temperature (Novick ). An entirely different genetic approach pioneered by Ray Deshaies, another one of my talented students, led to the discovery of the genes required for secretory and membrane protein insertion into the ER membrane (Deshaies and Schekman, 1987). In the years since these discoveries, >100 genes have been implicated in the yeast secretory pathway.

GENETIC AND MOLECULAR CLONING ANALYSIS OF SECRETORY MACHINERY

Novick and later postdoctoral fellow Chris Kaiser performed genetic and cytological analysis of single- and double-sec mutant cells to develop a temporal map of the secretory pathway (Novick ; Kaiser and Schekman, 1990). The map proved to be similar to the path charted by Palade for secretion of proteins in pancreatic acinar cells. Of course, the clear advantage of the genetic approach is that it allowed the identification of the genes and proteins required to operate the secretory pathway. Many of these genes were first identified in yeast, which allowed their mammalian homologues to be cloned and characterized.

Among the examples of striking evolutionary conservation of the SEC genes are SEC1, which encodes a regulatory protein required to control the activation of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins required for vesicle docking at multiple stages of vesicular traffic in all eukaryotic cells (Novick and Schekman, 1979); SEC17 and SEC18, which encode the yeast forms of the mammalian proteins NSF and α-synaptosome-associated protein involved in membrane vesicle docking and fusion (Kaiser and Schekman, 1990); SEC61, which encodes the major channel forming subunit of the secretory polypeptide translocase that is shared with the mammalian and bacterial equivalent function (Deshaies and Schekman, 1987); SEC4, which encodes a ras-like guanosine triphosphate (GTP) binding protein, the first to be implicated in the targeting of vesicles to target membranes (Salminen and Novick, 1987); SEC22, one of several ubiquitous SNARE proteins required to dock secretory vesicles (Kaiser and Schekman, 1990); and SEC21 and SEC23, two genes that encode subunits of distinct coat protein complexes that execute vesicle budding early in the secretory pathway (Hicke ; Hosobuchi ).

Yeast recombinant DNA technology also permitted the application of reverse genetics to the functional evaluation of proteins whose physiological role had not been established. Greg Payne, a postdoc in the lab, was the first to clone the gene for clathrin heavy chain that had long been assumed to play a role in secretion as well as in receptor-mediated endocytosis. Surprisingly, Payne discovered that yeast cells survived deletion of the clathrin heavy chain gene (Payne and Schekman, 1985), although one genetic background yielded an inviable null phenotype (Lemmon and Jones, 1987). Further characterization of viable null cells revealed a function for clathrin in the retention or retrieval of the mating pheromone processing endoprotease, Kex2p, in the trans-Golgi membrane (Payne and Schekman, 1989).

In the late 1980s, Hugh Pelham suggested that the major ATP-binding heat-shock proteins, hsp70, were involved in cytoplasmic protein refolding after denaturation (Pelham, 1986). A genetic test of hsp70 function became possible when Elizabeth Craig's lab characterized the hsp70 genes in yeast and found that one gene family, SSA1–4, encoded redundant essential functions. In collaboration with Maggie Werner-Washburne in her lab, Ray Deshaies discovered that hsp70 is required to present incompletely folded secretory and mitochondrial precursor proteins that engage in posttranslational translocation into the ER and mitochondrial membranes, respectively (Deshaies ). Gunter Blobel's lab reported the same requirement using a reconstituted secretory protein translocation reaction (Chirico ).

Before our work, two genetic studies in other organisms also yielded genes that control the secretory process. Secretion mutants defective in mucocyst discharge in Paramecium and Tetrahymena were isolated and characterized morphologically (Beisson ). However, the difficulty of molecular and genetic analysis in protozoa limited the potential impact of these early, fascinating studies. A landmark study on muscle uncoordinated mutants of Caenorhabditis elegans by Sydney Brenner yielded several mutants that were later found to affect neurosecretory processes (Brenner, 1974). The locus of one such mutant, unc-18, was molecularly characterized and found to encode a protein of significant homology to the first yeast Sec protein, Sec1p (Hosono ; Gengyo-Ando ).

BIOCHEMICAL DISSECTION OF SECRETORY MECHANISMS

Quite independently of our effort, a powerful biochemical approach was pioneered by James Rothman, whose laboratory developed a cell-free reaction to reconstitute traffic of a viral glycoprotein among compartments of the mammalian Golgi apparatus. Rothman's assays led to the isolation of a number of key proteins involved in vesicle biogenesis and fusion, including many that are encoded by the mammalian genes corresponding to the yeast SEC genes (Rothman, 2002).

The genetic approach did not immediately yield molecular mechanistic insights. Although it was clear that the yeast SEC genes had mammalian homologues, among the first group cloned only one, SEC4, showed a meaningful functional homology, in this case to the Ras GTP-binding protein superfamily (Salminen and Novick, 1987). This discovery, which launched Peter Novick on his independent career, presaged the identification of dozens of Rab proteins, each involved in targeting a vesicle to a unique intracellular destination.

My hunch all along was that a functional biochemical approach of the sort that Rothman's lab had established, would be necessary to bridge the gap between genetics and molecular mechanism. By the mid-l980s, David Baker in my lab and Hannele Ruohola (now Ruohola-Baker) in the lab of my former student Susan Ferro-Novick had developed a cell-free reaction that reconstituted the translocation and vesicle-mediated traffic of a secretory protein (Baker ; Ruohola ). The reaction faithfully reproduced the physiological event as evidenced by the reconstitution of sec mutant defects in vitro. Given the likely biochemical complexity of the full transport reaction, we focused on an initial step in the formation of vesicles that bud from the ER.

Michael Rexach exploited the reaction devised by David Baker to show that a gently prepared yeast lysate retains the ER in a rapidly sedimentable form but that slowly sedimenting vesicles are produced that capture secretory cargo from the ER lumen. Rexach and another student Linda Hicke showed that the sec mutants that Chris Kaiser had demonstrated to retard transport vesicle formation from the ER in vivo, do so in the cell-free reaction (Rexach and Schekman, 1991; Hicke ). Hicke, Christophe D'Enfert, Nina Salama, and Nancy Prior then purified the cytoplasmic proteins required for vesicle budding, and Charles Barlowe put them all together to discover they comprise a novel coat complex that we called COPII (Barlowe ).

In late 1990, I received a phone call from Lelio Orci whose morphological work on insulin maturation and collaborative effort with Jim Rothman on vesicle traffic in Golgi membranes reflected the highest standards of membrane ultrastructural analysis. He had some choice words about our published yeast morphology, but he offered his help, which I immediately accepted. He has been a valued colleague ever since that fateful call (Figure 1).

Figure 1.

Schekman and Orci planning a review article in Orci's office at the University of Geneva.

Further biochemical analysis by Ken Matsuoka, Bruno Antonny, and Marcus Lee revealed that the purified COPII coat assembly subunits are sufficient to initiate and complete the membrane shape changes that accompany vesicle budding and scission (Matsuoka ; Antonny ; Lee ). Chris Kaiser's lab and Yuval Shimoni in my lab found that one COPII subunit, Sec24p, confers cargo sorting specificity to COPII (Roberg ; Shimoni ). Liz Miller then mapped multiple cargo binding sites on Sec24p and presented the most direct evidence to date that membrane cargo sorting at the ER is achieved by selective interaction of the coat and cognate sorting signals on the cargo proteins (Miller ). Electron microscopic tomography and x-ray crystallographic analysis in the laboratories of William Balch and Jonathan Goldberg has now provided an atomic resolution image of the coat and how it assembles and captures membrane cargo during vesicle budding (Mossessova ; Stagg ; Fath ).

APPRECIATION AND OPPORTUNITIES

After an initial setback with my first National Institutes of Health (NIH) grant proposal, which was rejected because I lacked experience with yeast and had no preliminary data, my subsequent genetic and biochemical work on yeast was generously and continuously supported by National Institute of General Medical Sciences. Beginning in 1991, the Howard Hughes Medical Institute (HHMI) provided me an opportunity to extend this work in new directions, most recently into studies on membrane traffic in cultured mammalian cells (Fromme ; Kim ; Merte ). The added complexity and medical relevance of traffic in human cells will keep us busy for years to come.

I am grateful to the funding agencies (National Science Foundation, NIH, HHMI); to my academic home in the University of California; to my patient and understanding family; to my constant friends and collaborators Bill Wickner and Lelio Orci; and particularly to the many dedicated students, postdoctoral fellows, and loyal staff who have made my lab an exciting place to come to every morning (Figure 2)!

Figure 2.

Schekman's 50th birthday party and lab reunion at the 1998 ASCB meeting in San Francisco.