Cell, Vol. 25. 461-469, August 1981, Copyright 0 1981 by MIT Order of Events in the Yeast Secretory Pathway Peter Novick, Susan Ferro movement of labeled proteins from the endoplasmic and Randy Schekman reticulum (ER) to the cis face of the Golgi apparatus, Department of Biochemistry and from mature zymogen granules to the cell exterior University of California (Schramm, 1967; Jamieson and Palade, 1968b). Berkeley, California 94720 Other drugs that have a more selective effect on the secretory process, such as the ionophores A23187 and monensin, have been used to define stages in the Summary pathway further (Tartakoff and Vassalli, 1978). Re- construction in vitro of individual events, such as the The sequence of posttranslational events in the import of secretory proteins into the lumen of the ER export of yeast glycoproteins has been determined (Blobel and Dobberstein, 197.5) or the transport of a with the aid of mutants that affect the secretory membrane protein from the ER to the Golgi (Fries and apparatus. Temperature-sensitive secretory mu- Rothman, 1980) offers an analysis of this process at tants (set) of S. cerevisiae, when incubated at a the molecular level. nonpermissive growth temperature (37”C), accu- We have developed a genetic approach to the study mulate intracellular precursor forms of exported of the secretory process in Saccharomyces cerevi- glycoproteins, such as invertase, and expand or siae. Secretion in yeast is characterized by low levels amplify one or more of three different secretory of intracellular precursors, few secretory organelles organelles. Characterization of haploid double-sec- and a short lag time between synthesis and release at mutant strains, with regard to the structure of the the plasma membrane. These factors have hindered accumulated invertase and the morphology of the analysis of this process. The isolation of a large num- exaggerated organelles, allows assessment of the ber of conditionally lethal mutants that thermorever- order in which the gene products are required, the sibly trap secretory proteins at distinct steps in the sequence of invertase maturation steps and a path- pathway (Novick and Schekman, 1979; Novick et al., way of secretory organelles. The transitions from 1980) has enabled us to construct an assembly path- one organelle to the next require energy and set way leading from synthesis to release. gene products. One of the mutants (sec7) accumu- The use of double mutants in ordering the events in lates a different organelle depending on the con- a biosynthetic pathway was first described by Mitchell centration of glucose in the medium. In normal and Houlahan (1946) for the synthesis of adenine in growth medium (2% glucose), a thermally irreversi- Neurospora. Single mutants that block at phenotypi- ble structure, the Berkeley body, predominates; in tally distinguishable stages have been combined pair- low glucose (O.l%), Golgi structures accumulate wise and used to map pathways as complex as the thermoreversibly. The results are consistent with cell division cycle (Hartwell et al., 1974). Jarvik and the following model. Secretory proteins enter the Botstein (1973) developed a more sophisticated tech- ER, where the initial steps of glycosylation occur. nique for ordering events in the morphogenesis of Nine or more set gene products and energy are phage P22. By using reversible and independently required to transfer material to a Golgi-like struc- applied blocks, they demonstrated that the detection ture, where further glycosylation occurs. Two or of intermediate structures was not necessary for the more functions and energy are required to package assignment of a functional sequence of events. We nearly fully glycosylated proteins into vesicles that have used double-mutant analysis and a variation of are then transported into the bud, where they fuse the Jarvik and Botstein technique to order events in with the plasma membrane in a process that re- the yeast secretory pathway. quires at least ten additional gene products and energy. Results Introduction Secretion Is Rapid External invertase synthesis is derepressed by a de- The intracellular pathway followed by secretory pro- creased supply of glucose. The secreted enzyme re- teins in eucaryotic cells has been explored by several mains in the yeast cell wall and can be assayed in a techniques. Jamieson and Palade (1967a, 1967b) whole-cell suspension (Dodyk and Rothstein, 1964). used autoradiography and cell fractionation to follow The level of external invertase began to rise 10 min pulse-labeled secretory proteins through a series of after X2180 cells were transferred from YP medium membrane-bounded organelles. They found that tran- with 5% glucose to YP medium with 0.1% glucose sit of labeled proteins through the pathway was not (Figure 1). The specific activity increased at a constant blocked by cycloheximide, suggesting that ongoing rate for the next 30 min. During this period, inhibition protein synthesis does not force the flow of secretory of protein synthesis by cycloheximide allowed the products (Jamieson and Palade, 1968a). An inhibitor level of external invertase to rise for an additional 5 of oxidative phosphorylation, however, did block min as the internal precursor pool was depleted. This Cell 46.2 experiment suggested that inhibition of protein syn- vesicles, whereas a double mutant with sec7 elimi- thesis and completion of export require 5 min or less. nated only the large vesicles. Ontogeny of Secretory Organelles Maturation of lnvertase All but one of the mutants that accumulate secretory The set mutants have been divided into two classes enzymes also accumulate or exaggerate secretory based on the extent of glycosylation of the accumu- organelles (Novick et al., 1980). In most cases, only lated invertase (Esmon et al., 1981). Mutants that one of three different organelles accumulates in a accumulate ER produce a form of invertase that has mutant: ER, toroidal or cup-shaped structures called a shorter, possibly core, oligosaccharide. The other Berkeley bodies or 80-100 nm vesicles. If these or- mutants accumulate invertase that has the core and ganelles represent stages in the passage of secretory outer-chain oligosaccharide. These two forms can be proteins along a linear pathway, a double mutant distinguished by electrophoresis on SDS-polyacryl- should accumulate the organelle corresponding to the amide gels. The immature invertase migrates with an earliest block. apparent molecular weight of 79 to 83 kd; the mature A number of double set mutants were constructed form migrates diffusely with an apparent molecular in which each member, by itself, produced one of the weight of 100 to 140 kd. Double mutants were used three distinct phenotypes. Single and double mutants to evaluate the influence on invertase maturation. were examined by thin-section electron microscopy. Single and double set mutants (secl, sec78; sec7, Double mutants constructed from sec78 (ER and sec78) were grown in minimal medium and transferred some 40-60 nm vesicles), and sec20 (ER), secl (80- to 37°C under invertase derepressing conditions. 100 nm vesicles) or sec7 (Berkeley bodies), showed Cells were labeled for 1 hr with 35S042- and converted the sec78 phenotype (Table 1; Figure 2). Mutant to spheroplasts with lyticase (Scott and Schekman, alleles of all ten complementation groups that show 19801, and extracts were prepared by detergent lysis. 80-l 00 nm vesicle accumulation were combined with Labeled invertase was precipitated in a two-stage sec7, and in each case the double mutant accumu- reaction with antibody and fixed Staphylococcus A lated Berkeley bodies. Mutants with more complex cells. lmmunoprecipitates were dissolved in SDS and phenotypes were also examined in combination with were subjected to electrophoresis on polyacrylamide sec78 and sec7. Berkeley bodies and some 80-100 gels. Double mutants that included sec78 showed that nm vesicles are seen in sec74; a double mutant with this phenotype was epistatic to the secl and sec7 sec7 produced Berkeley bodies only. A mixture of form of invertase (Figure 3). In this experiment, the 80-l 00 nm and 40-60 nm vesicles, Berkeley bodies and ER is seen in sec79; combination with sec78 Table 1. Double-set-Mutant Phenotypes” prevented formation of Berkeley bodies and large Double-Mutant Phenotype with Single-Mutant 0.8. I I Phenotype sec7-7 secl8-1 Single-mutant phenotype Bbs ER and sv 0.6- S.SCl-1 ves Bbs ER and SY = sec2-56 ves and Bbs Bbs E 2 sec3-2 ves Bbs 2 0.4- sec4-2 ves Bbs 0 Shrft to 0.1% Glucose 5 sec5-24 ves Bbs is - Se&-4 ves Bbs set 7- 1 Bbs ER and sv sec8- 1 ves Bbs sec9-4 ves and Bbs Bbs 0 IO 20 30 40 set 7 O-2 ves Bbs Time (min) set 7 4-3 Bbs and ves Bbs Figure 1. Transit Time of lnvertase to the Cell Surface .9x75-1 ves Bbs X21 80-l A cells were grown overnight in YP medium with 5% glucose. secl9-1 ER and Bbs and Bbs and ER and ER and sv Cells (20 Asoo U) were sedimented in a clinical centrifuge, resus- vesand sv sv pended in 10 ml of YP medium with 0.1% glucose and incubated at 37°C. After 16 min, cycloheximide (final concentration 0.1 mg/ml) sec20- 1 ER ER and sv was added to a portion of the culture(~). At timed intervals 0.5 asv: small vesicles (40-60 nm). ves: vesicles (80-100 nm). Bbs: ml aliquots were chilled, sedimented in a clinical centrifuge and Berkelev bodies. resuspended in 0.5 ml of 10 mM NaN3 at 0°C. Yeast Secretory Pathway 463 Figure 2. Thin-Section Electron Micrographs of Cells Grown in YPD Medium (A) HMSF 163 (sec22-I) grown at the permissive temperature (25°C). (B) SF 230-I (secl-1, secl&1) grown for 2 hr at 37°C.