Ultrastructural modifications of vesicular and Golgi elements in the Saccharomyces cerevisiae sec21 mutant at permissive and non-permissive temperatures.код для вставкиСкачать
THE ANATOMICAL RECORD 240:32-41 (1994) Ultrastructural Modifications of Vesicular and Golgi Elements in the Saccharomyces cerevisiae sec21 Mutant at Permissive and Non-Permissive Temperatures A. RAMBOURG, Y. CLERMONT, C.L. JACKSON, A N D F. KfiPkS Departement de biologie cellulaire et moleculaire du CEA, Centre &etudes de Saclay, France (A.R., F.K., C.L.J.); Department of Anatomy and Cell Biology, McGill University Montreal, Quebec, Canada (Y.C.) ABSTRACT Background: The secretory protein transit between cisternae of endoplasmic reticulum (ER) and Golgi elements is blocked when the yeast Saccharomyces cerevisiae sec21 mutant is shifted from the permissive (24°C) to a non-permissive (37°C) temperature, but 30-50 nm vesicles accumulate in the cytoplasm. At the semi-permissive temperature of 33°C there is no complete block but rather a slowdown of the protein transport between ER and Golgi. The purpose of the present investigation is to analyze the structural expression of these events. Methods: S. cerevisiue sec21 mutants were maintained for 90 min at semirestrictive (33°C) or restrictive (37°C) temperatures and then progressively returned to 24°C. Following fixation in glutaraldehyde and a postfixation in potassium ferrocyanide reduced osmium, 0.08 to 0.2 pm thick sections were cut from Epon embedded yeasts. Using the thicker sections, stereopairs of electron microscope photographs were prepared and used to visualize the three-dimensional configuration of the organelles. Results: At permissive temperature, the Golgi elements appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm. The diameter of these membranous tubules varied considerably from one Golgi element to another. Larger tubules showed at their intersections distensions with size and staining intensity comparable with that of the secretory granules seen at proximity of the Golgi networks or at the cell periphery. Small vesicles in the 30-50 nm size range were rarely if ever observed in cells grown at permissive temperature. Golgi networks and secretion granules were less conspicuous in mutant cells maintained at 33°C and completely disappeared at 37°C. In both cases, the main structural feature was the presence in the cytoplasm of numerous small vesicles and of short membranous tubules with a diameter identical to that of the small vesicles. As soon as 5 minutes after shifting mutants from 33°C to 24"C, the small vesicles disappeared from the cytoplasm, while secretory granules were actively produced in extensively developed Golgi network. When mutants were returned from 37°C to 24"C, the disappearance of small vesicles was more progressive and concomitant with the progressive reconstruction of Golgi networks. Conclusions: It is thus postulated that, in the above mentioned conditions, the small vesicles of the see21 mutant did not act as intermediate carriers between the endoplasmic reticulum and a pre-existing Golgi apparatus, but rather fused together to produce newly formed Golgi networks. o 1994 Wiley-Liss, Inc. Key words: Secretion pathway, Yeast cells, Carrier vesicles, Golgi apparatus In the yeast Saccharomyces cerevisiae, the Golgi apparatus consists Of saccules Or cisternae distributed throughout the cytoplasm- This was observed with the electron microscope in freeze fracture replicas 0 1994 WILEY-LISS, INC Received January 24, 1994; accepted May 2, 1994. Address reprint requests to Dr. A. Rambourg, Dbpartement de biologie cellulaire et mol6culaire, Centre d'6tudes de Saclay, Gif-surYvette 91191 Cedex France. ULTRASTRUCTURAL MODIFICATIONS IN SECZl (Svoboda and Necas, 19871, in thin sections of yeasts fixed with potassium permanganate (Kaiser and Schekman, 1990), or in sections immunocytochemically stained with specific markers of the Golgi apparatus (Preuss et al., 1992).When the three-dimensional configuration of this organelle was examined in stereopairs of thick sections of glutaraldehyde fixed cells postfixed with reduced osmium, the Golgi apparatus appeared as isolated networks of membranous tubules that were readily distinguished from the less intensely stained cisternae of the endoplasmic reticulum (ER) (Rambourg et al., 1993). Furthermore, in wild type cells, small vesicles (30-50 nm in diameter) was rarely if ever seen next to the Golgi elements or elsewhere in the cytoplasm (Rambourg et al., 1993). In mammalian cells, equivalent small vesicles seen in the Golgi region are said to serve in the transport of proteins from the ER to the cis-face of the stacks of Golgi saccules or from one saccule to the next in the cis-trans direction (reviews Farquhar, 1985; Rothman, 1985; Rothman and Orci, 1992; Mellman and Simons, 1992; LippincottSchwartz, 1993). In S. cerevisiae secl7, secl8, and sec22 mutants, in which secretory protein transport between the ER and the Golgi elements is disrupted when they are transferred from a permissive (24°C) to a non-permissive temperature (37"C),small vesicles, 50 nm in diameter, accumulate in the cytoplasm (Kaiser and Schekman, 1990). The S. cerevisiae sec21 mutant is also deficient in protein transit from the ER to the Golgi apparatus when shifted to a non-permissive temperature, but accumulates 30-50 nm vesicles but in smaller numbers than in the above mentioned mutants (Kaiser and Schekman, 1990). As determined by pulse chase analysis after the in vivo labeling of sec21 cells and immunoprecipitation of carboxypeptidase Y (CPY), an enzyme which migrates toward the vacuole through the secretory pathway, the sec21 mutant accumulated exclusively the core glycosylated p l precursor form of the enzyme when shifted to the non-permissive temperature of 37°C (Hosobuchi et al., 1991). This result indicated that under these conditions there was a complete block of the ER to Golgi transport of CPY. However, when this mutant was shifted to the semi-permissive temperature of 33"C, there was a partial accumulation of the peripherally glycosylated p2 precursor form and the mature vacuolar form was also present. It was therefore proposed that at 33°C there was no block in the ER to Golgi transport but rather a slow-down, within the Golgi elements, of the migration of the enzyme en route toward the vacuole (Jackson and Kepes, 1994). . The purpose of the present investigation is to establish the structural expression of these events and more precisely to describe the modulations of the Golgi and vesicular elements when sec21 mutants were shifted for various time intervals to semi-restrictive (33°C) or restrictive (37°C)temperatures and then progressively returned to the permissive temperature of 24°C. 33 1989).The mutant strain (sec21) was RSY277 (sec21-1, ura3-52, MATor). Yeast strains were usually grown at 24°C in YPD medium (2% Bacto-peptone, 1%Bacto-yeast extract, 2% glucose). Culture media were obtained from Difco Laboratories (OSI, Paris). Cell density was monitored in liquid cultures by measuring optical density a t 600 nm (OD 600) using a DU 68 spectrophotometer (Beckman Instruments France, Gagny). Cells that were examined by electron microscopy came from exponentially growing cultures grown in a shaking water bath and maintained below an OD 600 of 0.5. Non-permissive and semi-permissive temperatures for the thermosensitive sec21 mutant were 37 and 33"C, respectively. Cells were either maintained a t 24"C, or shifted for 90 min to the higher temperature, and shifted back to 24°C for 0, 5, 10, 20, or 40 min. Cell fixation was initiated by adding 2% glutaraldehyde directly to the culture while maintaining the growth conditions during 5 min. Cells were then harvested and resuspended overnight at room temperature in a fixative containing 2% glutaraldehyde in 0.1M cacodylate buffer, pH 6.8, and 0.8M sorbitol. After centrifugation, cells were resuspended and postfixed for 1 hr at room temperature in a 1:l mixture of 2% aqueous osmium tetroxide and 3% aqueous potassium ferrocyanide (Karnovsky, 1971). Dehydration was carried out in ethanol followed by embedding in Epon. Point zero eight to 0.2 pm thick sections were cut with a Reichert automatic ultramicrotome and counterstained for 2 min with lead citrate prior to examination with a CM 12 Philips electron microscope a t 80 KV. For stereoscopy, grids were placed on the goniometric stage of the electron microscope, and stereopairs were obtained by taking pictures of the same field after tilting the specimen at -15" and +15" from the 0" position. A three-dimensional magnified image of the structures was obtained by looking at properly adjusted pairs of such photographs with a stereoscopic binocular lens. RESULTS Wild Type Strain and Sec21 Mutant at 24°C In the wild type strain of S. cerevisiae as well as in sec21 mutant cells grown at the permissive temperature of 24"C, the cytoplasm contained relatively few ER cisternae and Golgi elements (Figs. 1-4). The cell wall and the vacuole(s) were heavily stained following reduced osmium postfixation, whereas the nuclear content and mitochondria were unstained (Figs. 1-3). The endoplasmic reticulum, the content of which showed an intermediate staining, consisted of poorly fenestrated sheets or cisternae connecting the nuclear envelope and extensive subplasmalemmal cisternae (Figs. 1, 2). More intensely stained networks of anastomosed membranous tubules corresponded to Golgi elements (Figs. 2, 3). The diameter of these membranous tubules varied considerably from one Golgi element to another or along the same network (Figs. 2, 3). Some Golgi eleMATERIALS AND METHODS ments showed thin tubules and relatively small distenTwo Saccharomyces cerevisiae strains were used sions at the branch points (Fig. 2). Other Golgi elements showed distended tubules with large dilations at throughout these studies: the wild type strain (SEC + was W303 1B (leu2-3, -112 ade2-1 ura3-1 his3-11, the junctions of the meshwork (Figs. 2, 3). Such dila-15 trpl-1 can1 -100 MATor, Rothstein and Thomas, tions showed a size and staining intensity comparable 34 A. RAMBOURG ET AL. to that of the secretory granules often seen a t proximity of these Golgi networks or close to the plasma membrane (Figs. 1, 3, 4) in particular in the region of the bud whenever a bud was present. Golgi elements also showed interconnected tubules that were of a size intermediate to that of the two networks described above. Some of these Golgi elements were closely apposed to the nuclear envelope or the other ER cisternae (Figs. 1, 2). Characteristically small vesicles in the 30-50 nm range were generally absent or exceedingly rare within the cytoplasm of these cells (Figs. 1-4). Sec21 Mutant at the Semi-Restrictiveand Restrictive Temperatures When see21 mutant yeast cells were shifted for 90 min to semi-restrictive (33°C) and restrictive (37°C) temperatures, the staining characteristics and morphological features of the ER were identical to those of controls. In contrast, the structure of the Golgi elements and the number of secretory granules were modified and varied depending on the temperature t o which the cells had been shifted. Thus at 33"C, the Golgi networks were less conspicuous than in controls and whenever present they were made up of fine anastomosed tubules (Figs. 5, 7). In other words, the Golgi elements formed by a network of large tubules were virtually absent. However, secretory granules although rare were still present (Figs. 5-7) and seen in the regions of the buds. At this temperature the main structural feature observed was the presence of numerous small vesicles in the 30-50 nm size range and short membranous tubules with a diameter identical to that of the small vesicles (Figs. 6, 7). A t 37°C the networks of tubules forming the Golgi elements were generally absent, but, whenever present, they were composed of fine disorganized tubules (Figs. 8, 9). Secretory granules were absent while the 30-50 nm vesicles and short fine tubules were abundant (Figs. 8,9). Sec21 Mutants First Maintained at 33°Cor 37°C and Returned to the Permissive Temperature of 24°C In cells maintained for 90 min a t 33°C and then returned for 5 min to 24°C there was a striking diminution, if not a complete disappearance, of small 30-50 nm vesicles and of short fine membranous tubules (Fig. 10). At this or longer time intervals after the temperature shift (i.e., 10,20 min), the Golgi elements formed of fine or large nodular anastomotic tubules became more conspicuous and even more abundant than in controls (Figs. 11, 12). Not infrequently such Golgi elements were closely apposed to ER cisternae (Fig. 13). Strongly stained secretory granules reappeared (Figs. 10-12). At 40 min after the shift to the permissive temperature the structural features of the cells were identical to that of controls (Fig. 13). In cells maintained for 90 min at 37°C and then returned to the permissive temperature of 24"C, Golgi networks of various sizes and abundant secretory granules were present as soon as 5 min after the temperature shift (Figs. 14, 15). However, a t this time interval some small vesicles and tubules, 30-50 nm in diameter, were still present in the cytoplasm (Figs. 15, 16). At longer time intervals, i.e., a t 10, 20, or 40 min, the small vesicles and fine short tubules disappeared and the morphological appearance of the cells was returned to that of controls (Figs. 17, 18). DISCUSSION Structure of Golgi Elements in Sec2l Mutant at Permissive Temperature The yeast S. cerevisiae see21 mutant, maintained a t a permissive temperature, like wild type cells, contains several Golgi elements widely dispersed throughout the cytoplasm (Svoboda and Piedra, 1983; Svoboda and Necas, 1987; Makarow, 1988; Preuss et al., 1992). These elements, when examined in electron microscope, stereoscopic images of thick sections appear as isolated tubular networks (Rambourg et al., 1993) (Fig. 19). This is in contrast to most mammalian cells in which the Golgi apparatus is composed of stacks of saccules (reviews Farquhar and Palade, 1981; Goldfischer, 1982). Such stacks are usually connected to each other to form a single organelle (Rambourg et al., 1974; Rambourg and Clermont, 1990). Furthermore, the Golgi elements of yeast, which stain more intensely with reduced osmium than the ER cisternae, often show dilations at the intersections of the interconnected tubules that are similar in size and staining properties to the secretion granules. Hence, it has then been postulated that in yeast, secretion granules are formed by rupture of these tubular networks (Rambourg et al., 1993), considered as equivalent to the trans-tubular or trans-Golgi network of mammalian cells (Rambourg et al., 1979,1987; Roth et al., 1985; Griffiths and Simons, 1986).However, it was noted in the present study that, in addition to these relatively large dilations, there were also in see21 mutants smaller dilations at the intersections of membranous tubules the calibre of which was obviously smaller than that of the tubules of the so-called trans-tubular networks. These networks of small membranous tubules were preferentially located in the vicinity of the endoplasmic reticulum and were occasionally continuous with the networks of larger tubules, thus suggesting that the former might tentatively be considered as precursors of the latter. Presence of Small Vesicles in Sec21 Mutants at Restrictive Temperatures It should be mentioned that in wild type S. cerevisiae, as well as in sec7 mutants (Rambourg et al., 1993) or in see21 mutants grown a t a permissive temperature, small 30-50 nm vesicles similar to those observed in association with the Golgi apparatus of mammalian cells (Farquhar and Palade, 1981; Orci et al., 1986, 1989) were exceptionally, if ever, encountered. When see21 mutants grown a t a permissive temperature are shifted to a semi-restrictive temperature (33"C), small vesicles appeared within the cytoplasm while the structure of the fine Golgi networks and the production of secretion granules were hardly affected (Fig. 19). This observation is in keeping with the proposal commonly accepted that small vesicles may represent transport intermediates between the endoplasmic reticulum and the Golgi apparatus (Palade, 1975; Novick et al., 1981; Paulik et al., 1988; Kaiser and Schekman, 1990) and might therefore accumulate in conditions producing a slow-down in the transport of secretory proteins, as it is the case for see21 mutants maintained a t 33°C (Jackson and K6pBs, 1994). Figs. 1, 2. Stereopairs of 0.1 p thick sections of portions of S. cerevisiae sec21 mutant maintained at the permissive temperature of 24°C. These preparations, as well as all those of the present study, show a well stained cell wall and Golgi elements, less intensely stained cisternae of the endoplasmic reticulum, and nuclear envelope. The vacuole (V) shows a variable staining, while mitochondria (m) and nuclear content (N) are unstained. For each stereopair a single magnified stereoscopic image may be obtained by utilizing a properly adjusted (at 65 mm) binocular lens. Fig. 1. Proximal to nuclear envelope a side view of a Golgi element is indicated (open arrow). Secretion granules are seen next to the cell surface (arrow). Arrowhead indicates a n ER cisterna bridging the nuclear envelope and a subplasmalemmal cisterna. Note the absence of small 30-50 nm vesicles. X 22,000. Fig. 2. Several Golgi elements under the form of networks are visible. The one above in contact with a n ER cisterna (arrow), is made up of relatively large membranous tubules. The one below is made up of small tubules (arrowhead). V, vacuole. x 22,000. Fig. 3. Portions of two adjacent yeast cells both showing Golgi networks face view and side view (small arrows in the cell above) or a network of nodular tubules (arrowhead in the cell below). The latter shows continuity with a subplasmalemmal ER cisterna. Note the absence of small 30-50 nm vesicles. N, nucleus; m, mitochondrion; V, vacuoles. x 28,000. Fig. 4. Small portion of a yeast cell. Golgi elements are identified seen side view (arrow above) or face view (open arrow). Several secretory granules are visible (arrowheads). ER, ER cisternae; F, fenestrated spherule; V, vacuole. x 28,000. Figs. 5-7. Stereopairs of S . cereuisiue sec21 mutant maintained for 90 min at the semi-permissive temperature of 33°C. x 22,000. Fig. 5. The nuclear envelope and ER cisternae (small arrows) and a few secretory granules are seen (S). In addition an extensive irregular network of Golgi tubules is visible (open arrow). It is made up of relatively small tubules. Fig. 6. In addition to usual organelles, i.e., ER cisternae (arrows), nuclear envelope delimiting the nucleus (N), mitochondrion (m), and secretion granules (S); small 30-50 nm vesicles were also observed (arrowheads). A small fenestrated sphere (F) is also indicated. Fig. 7. In addition to a fine Golgi network (open arrow) numerous small vesicles in the 30-50 nm range (arrowheads), as well as short tubules of small caliber (small arrows) are distributed throughout the cytoplasm. V, vacuole; N, nucleus. Pale stained ER cisternae are also indicated (ER). ULTRASTRUCTURAL MODIFICATIONS IN SECZI 37 Figs. 8, 9. Stereopairs of S. cereuisiae see21 mutant maintained for 90 min a t the nonpermissive temperature of 37°C. x 22,000. trated sphere; cisterna of endoplasmic reticulum, ER, aggregate of material of unknown nature (white asterisk). Fig. 8. The cytoplasm contains several small 30-50 nm vesicles, and short small membranous tubules (small arrows). A disorganized small Golgi tubular network is indicated (open arrows). N, face view of the nuclear envelope showing nuclear pores; V, vacuoles; F, small fenes- Fig. 9. In addition to some small 30-50 nm vesicles (arrowheads) a loose fragmented network of fine Golgi tubules is indicated (open arrow). V, vacuole; N, nucleus; ER cisternae (small arrows). When, however, these mutants were shifted to a nonpermissive temperature (37"C), the situation was different. As expected, secretion granules were no longer produced and Golgi networks were no longer observed within the mutant cells. Yet, in contrast to what is shown to occur in mutants such as secl8 (Kaiser and Schekman, 1990), not only vesicles but also small tubules and fragments of networks of the small variety were interspersed within the cytoplasm (Fig. 19). These observations suggest the possibility that such vesicles and tubules might result from the fragmentation of pre-existing Golgi networks. A final possibility was suggested by the kinetics of ultrastructural modifications occurring during return of sec21 mutant cells to a permissive temperature. As soon as 5 minutes after shifting the mutants from a 33°C to 24"C, the small vesicles had disappeared from the cytoplasm and secretion granules appeared to form actively from extensively developed Golgi networks. The reappearance of secretion granules and Golgi networks was instead more progressive when the yeast cells were returned from 37°C to 24°C (Fig. 19). After 5 minutes, the cytoplasm, which still contained numerous vesicles and tubules, exhibited rudimentary Golgi networks and the reappearance of clearly delineated networks at 10 minutes was concomitant with the dis- 38 A. RAMBOURG ET AL. Fig. 10. Photograph of a S. cereuisiae sec21 mutant initially maintained for 90 min at 33°C and then shifted for 5 min a t the permissive temperature of 24°C. Extensive Golgi tubular networks are seen in the cytoplasm (open arrow). They are of several varieties and composed of medium or small anastomosed tubules (above) or of tubules of larger size (below). A secretion granule is indicated (arrowhead). Small vesicles or short fine tubules are absent. N, nucleus; V, vacuole; ER, cisternae of ER. x 35,000. Fig. 11. Photograph of a small portion of a S. cereuisiae sec21 mutant initially maintained for 90 min at 33°C and then shifted for 10 min at 24°C. Next to the nucleus (N) there is a n extensive network of Golgi tubules of the larger caliber (open arrow). Such elaborate networks were not observed in controls. Secretory granules are also present (arrowhead). x 35,000. appearance of the tubules and small vesicles. It is therefore proposed that, under these conditions, the small vesicles did not act as intermediate carriers between the endoplasmic reticulum and a pre-existing Golgi apparatus but rather appeared to fuse together to give rise to newly formed Golgi networks. A rapid fusion of tubules and vesicles to form such networks Fig. 12. Photograph of a portion of a S. cereuisiue sec2l mutant maintained for 90 min at 33°C and then shifted for 20 min at 24°C. Next to a grazing section of the nuclear envelope (N) there are several Golgi networks made up of relatively large tubules (open arrows). Secretory granules are also abundant (arrowheads). m, mitochondrion. x 35,000. Fig. 13. Photograph of a portion of a S. cereuisiae sec21 mutant maintained for 90 min at 33°C and then shifted for 40 min at 24°C. At proximity of an ER cisterna (ER) there are networks of small and large Golgi tubular networks (open arrow) and secretion granules (arrowhead). Small vesicles are absent. N, nucleus. x 35,000. might perhaps explain the scarcity of vesicles in wild type cells or in sec21 mutants grown at a permissive temperature. ACKNOWLEDGMENTS The work done at McGill University was supported by a grant of the Medical Research Council of Canada. Figs. 14-16. Photograph of portions of S. cereuisiue sec21 mutant maintained for 90 min at 37°C and shifted for 5 min at 24°C. x 35,000. Fig. 14. In addition to ER cisternae (ER), nucleus (N), and vacuoles (V) the cytoplasm contains some Golgi networks of various sizes (open arrow) and a few secretory granules (small arrows). Fig. 15. Several secretory granules (arrowheads) close to the cell surface. A few small vesicles in the 30-50 nm range are indicated (small arrows). ER, ER cisterna. Fig. 16. In the cytoplasm of this cell there are numerous small 30-50 nm vesicles (small arrows) and a few secretory granules (arrowheads). N, nucleus; ER, ER cisternae; V, vacuole. Figs. 17, 18. Photographs of portions of S. cereuisiae sec21 mutant maintained a t 37°C for 90 min and then shifted for 10 min at 24°C. x 35,000. Fig. 17. In the cytoplasm of this cell there is a n exceptionally large network of large anastomotic tubules (open arrow). Secretory granules are also visible at proximity (arrowheads). N, nucleus; ER, peripheral ER cisterna. Small vesicles are absent. Fig. 18. In addition to the nucleus (N) and ER cisternae (ER) the cytoplasm contains a Golgi element composed of large anastomosed tubules (open arrow). As in Figure 17, the cytoplasm does not contain the 30-50 nm vesicles. 40 A. RAMBOURG ET AL. Fig. 19. Diagram summarizing the observations made on ER cisternae (ER), Golgi elements (GI, and secretory granules (SG) of S . cereuzsiae sec21 mutant maintained a t permissive, semi-permissive, and non-permissive temperatures. N, nucleus delimited by a porous nuclear envelope; CW, cell wall; V, small vesicles in the 30-50 nm size range; T, short tubules with a diameter identical to that of the small vesicles. A Sec21 mutant maintained at the permissive temperature of 24°C which serves as control. The Golgi elements appear as tubular networks. Secretion granules which appear to form by the breakdown of the larger anastomotic Golgi tubules accumulate in the cytoplasm facing the growth bud. Small 30-50 nm vesicles are rare. B: Sec21 mutant maintained at the semi-permissive temperature of 33°C. The Golgi elements are made up mainly of fine anastomotic tubules. A few secretory granules are present. Small vesicles become more numerous. C Sec21 mutant maintained at the non-permissive temperature of 37°C. Golgi elements formed of larger anastomotic tubules as well as secretion granules are absent. Some Golgi elements made up of fragmented networks of fine tubules are visible while short membranous tubules and small vesicles are abundant. D Sec21 mutant maintained at the non-permissive temperature of 37°C for 90 min then for 20 min a t 24°C. Golgi elements appear as elaborate networks of large nodular anastomotic tubules, more extensive than in controls. 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