- Email: [email protected]
Organelle inheritance in a test tube: the yeast vacuole
seminars in
CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 517–524
Organelle inheritance in a test tube: the yeast vacuole Albert Haas and William Wickner
these organelles in vivo and in vitro;7,9-13 (iii) several marker proteins for vacuoles have been characterized and their genes cloned,7 making vacuole analysis easier; (iv) powerful genetic tools are available for the study of S. cerevisiae and it can be readily grown in large quantities using simple methods.
Vacuoles (lysosomes) of the budding yeast Saccharomyces cerevisiae are low copy-number organelles which are inherited from mother to daughter cells. We are interested in the molecular basis of the division and segregation of vacuole material and in the coordination of these events with the cell cycle. Using microscopic, genetic and biochemical methods, we have shown that vacuole inheritance is a regulated process, described several genes and gene products involved in the course of inheritance, and developed a cell-free system which allows an in-vitro reconstitution of events that occur during vacuole inheritance in vivo.
The classical morphological approach: vacuole inheritance in vivo Initial microscopic studies showed that the growing bud receives most of its vacuolar material from the mother cell.9 Further analysis, using yeast zygotes, demonstrated that there is a ‘communication’ of vacuolar material between the two mating partners.12 Certain ‘tracks’ clearly exist in a zygote which conduct vesicles derived from either parental cell vacuole. These vesicles establish the vacuole in the emerging bud. At the same time, these tracks always run from parental vacuoles into the bud, never directly between the vacuoles from the mating partner (a- and α-) cells.12 Further studies showed that specific tracks are also used for the transport of vacuolar vesicles when haploid yeast cells were examined.14,15 Obviously, vesicular and tubular ‘segregation structure’ formation from the mother vacuole is coordinated by the cell cycle (occurring between S phase and G2/M phase transition) and segregation structure transport into the bud is spatially restricted to these direct connections (Figure 1). As the last discernible step of vacuole inheritance, the segregation structures fuse, allowing both mother and separated daughter cells to possess one or a few vacuoles (Figure 1; ref 14).
Key words: membrane fusion / organelle inheritance / vacuole / yeast ©1996 Academic Press Ltd
CYTOPLASMIC ORGANELLES are not produced de novo after cell division but arise from the growth, multiplication and separation of preexisting organelles. High copy-number particles, such as ribosomes, may be inherited by random diffusion. Inheritance of low copy-number organelles, however, requires a cell cycle-regulated multiplication and separation processes to ensure a proper partitioning between mother and daughter cells.1,2 In the budding yeast Saccharomyces cerevisiae, the inheritance of the nucleus,3 endoplasmic reticulum,4 Golgi apparatus,5 and mitochondria6 have been analysed. However, we are only at the start of studies of the molecular mechanisms underlying organelle multiplication and partitioning. This laboratory is interested in the analysis of the molecular mechanisms which enable baker’s yeast to faithfully inherit vacuoles7,8 during cell multiplication. We have chosen this organism and this organelle for practical reasons: (i) Vacuoles are very large lowcopy organelles and therefore can be observed by phase-contrast optics;9 (ii) specific fluorophores are available which accumulate in the acidic, esterase-rich vacuolar lumen and can be used to readily identify
The genetic approach: temperature-sensitive inheritance mutants The analysis of temperature-sensitive yeast mutants in secretion (sec mutants) or in vacuole protein sorting (vps mutants) have profoundly influenced our understanding of these pathways.7,16-18 The temperaturesensitive phenotype allows for the isolation of the respective genes by complementation of the mutated
From the Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3840, USA ©1996 Academic Press Ltd 1084-9521/96/040517 + 08 $18.00/0
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A. Haas and W. Wickner sensitivity of the strains to calcium, lysine, histidine or calcium, defects in sulfur metabolism, or screening for vacuole protease defects. The same gene (VP33) can therefore have several aliases, here CLS14, MET27, PEP14, SLP1, VPT33 and VAM5 (see ref 26).
locus. Analysis of mutants for a defect in vacuole morphology, however, is more difficult because of the lack of a phenotype which controls cell growth. Only about 50% of all mutants without a vacuole are temperature-sensitive.19,20 Therefore, the tedious microscopic screening of temperature-sensitive mutants,21 or a screen based on the somewhat higher density of cells lacking vacuoles,22 or survival in the presence of chloroquine23 were used to isolate vacuole inheritance (vac) mutants. Also, pink (or white) derivatives of otherwise red ade-2 yeast colonies showed an increased incidence of vac mutations providing a colour screening procedure.23 The analysis of vac mutants has yielded important insights. It has been established that vacuole inheritance and vacuole protein sorting can be studied separately. Some vac mutants, like vac1-1 ( = vps19),24,25 missort proteins which are normally delivered to the vacuole and, at the same time, fail to deliver vacuoles to the bud at non-permissive temperatures. The vac2-1 mutant, however, has a vacuole inheritance defect but no protein sorting defect.21 Interestingly, some vacuole morphology mutants, such as vps33, were identified by several independent screens, such as vacuole protein missorting, hyper-
The in vitro biochemical approach: taking apart the subreactions This approach is based on the classic reductionist approach of enzymology. It assumes that complex biochemical reactions which occur in the cell can also occur in a cell-free environment under the proper conditions. Molecular analysis then becomes feasible, as the environment can be shaped by adding or omitting isolated factors. In fact, there are several examples of successful reconstitutions of intracellular trafficking reactions.27 As a first step to a biochemical dissection of vacuole inheritance processes, we have employed semi-intact yeast cells.14 These are spheroplasts that have been permeabilized through a combination of osmotic shock and slow freezing.28 They retain much of their organellar architecture, yet are permeable to low-
Figure 1. Vacuole morphology during the cell cycle of S. cerevisiae. The top portion shows a schematic representation of the events recorded in the photographs in the lower portion. Yeast cells were labeled in vivo using the fluorogenic, vacuole-staining dye CDCFDA and observed by fluorescence microscopy. The phases of the cell cycle represented by the cells shown are indicated. (Reproduced from Conradt et al, The Journal of Cell Biology, 1992, vol. 119, pp. 1469-1479 by copyright permission of The Rockefeller University Press, ref 14.)
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Vacuole inheritance in vitro ‘fusion’ reactions we are observing require ‘upstream’ components of the inheritance apparatus (e.g. priming or vesiculation factors). Our cell-free system requires proteins which are sensitive to pharmacological reagents such as mastoparan (a G-protein activator), GTPγS (which is only very slowly hydrolysed by GTPases), neomycin (which binds strongly to polyphosphoinositides), bafilomycin (an inhibitor of vacuolar proton-pumping ATPases), and microcystin-LR and okadaic acid (inhibitors of serine/threonine protein phosphatases). Using these inhibitors,14,29,30 we could show that the overall reaction in vitro actually consists of several steps. Four subreactions have been defined: first, the isolated vacuoles must be briefly in contact with physiological salt concentrations; second, they must be incubated with cytosolic components; third, vacuoles react with added ATP, and fourth, the final stages of the reaction require incubation of vacuoles at a minimal vacuole concentration and a physiological temperature. The fusion reaction requires these steps in precisely this sequence.30 Although the question remains whether
molecular weight substances and to macromolecules, such as soluble proteins. Segregation structure formation in such cells, which lack much of their soluble components, was dependent on the external addition of energy (ATP) and was stimulated by the addition of a preparation of cytosol. These reactions did not occur, however, when vac1 or vac2 cells and cell components were used in the experiments, underscoring the authenticity of the reactions observed.14 These data suggested that reconstitution of vacuole inheritance reactions in a cell-free system should, in principle, be feasible. In the next step, we have therefore analysed vacuole information reactions using isolated components. Isolated vacuoles not only form segregation-like structures, but also fuse in the test tube when they are incubated with cytosol, an ATP-regenerating system and a physiological salt mixture at a physiological temperature (25 to 30°C).14 However, when components of the in-vitro reactions were isolated from vac mutants, the vacuoles fragmented and/or would not fuse.10,14,29 This close correlation between in-vivo and in-vitro observations allowed us to conclude that the reactions seen in the test tube are authentic reconstitutions of reactions occuring in the living cell. Encouraged by this reconstitution of at least two steps of vacuole inheritance (structure formation and vacuole fusion) in a cell-free system, we established a modified in-vitro assay which would allow us to quantify fusion frequency biochemically rather than by microscopic observation of vacuole morphology and size (Figure 2). The basis for this system is the observation that vacuoles possess a membrane-bound alkaline phosphatase (Pho8p) which reaches into the vacuolar lumen and which is imported as an enzymatically inactive precursor (proPho8p). In the vacuole lumen, proPho8p is cleaved by the vacuolar proteinase A (Pep4p), and thus gains alkaline phosphatase activity. Therefore, the vacuoles used in the standard cell-free reaction are isolated from two yeast strains, one with a deletion in the PHO8 gene, the other with a deletion in the PEP4 gene. Neither vacuole population has alkaline phosphatase activity. Enzymatic activity can only be generated by the mixing of the luminal contents of both vacuole types, and hence is a measure of frequency of vacuole fusion in vitro (Figure 2 and 3; ref 29). This in-vitro vacuole fusion reaction is again defective when components are used from vac-1, vac-2 or vac-5 cells (refs 10,14,29,29a), and therefore represent physiologically authentic reactions. Also, studying components from several vac cells in our cell-free reactions, it became clear that the
Figure 2. Scheme describing the pro-alkaline phosphatase maturation assay which is routinely used to quantitate homotypic vacuole fusion. For details see text. (Reproduced from Haas et al, The Journal of Cell Biology, 1994, vol. 126, pp. 87-97 by copyright permission of The Rockefeller University Press, ref 29.)
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A. Haas and W. Wickner the identical events occur in intact yeast cells, this study nevertheless allows for the isolation of stagespecific factors. One of the particular advantages of a cell-free system is the possibility of using specific antibodies to probe for the involvement of certain factors in the
reaction. Yeast cells mutated in the ras-like GTPase Ypt7p have fragmented vacuoles31 (Figure 4). Using specific Ypt7p-antibodies, we could show that this protein is absolutely required for homotypic (self-) vacuole fusion in both, the microscopic fusion assay (Figure 5) and the pro-Pho8p maturation assay
Figure 3. Kinetics of cell-free vacuole-to-vacuoles fusion. All parts of this figure are from the same experiment. (A) A mixture of vacuoles from pho8 and pep4 strains was incubated with cytosol, an ATP-regenerating system and salt at 25°C. Vacuoles fuse over time, as seen by a decrease in vacuole number and an increase in their average size. The vacuoles have been stained with a vacuolespecific fluorphor. (B) In parallel, the maturation of proPho8p was analysed by immunoblotting reaction samples removed at various times. (C) At various times, samples were removed and their alkaline phosphatase activities determined. These parallel experiments clearly demonstrate that generation of alkaline phosphatase activity parallels the enlargement of vacuoles and therefore represents homotypic vacuole fusion. (Reproduced from Haas et al, The Journal of Cell Biology, 1994, vol. 126, pp. 87-97 by copyright permission of The Rockefeller University Press, ref 29.)
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Vacuole inheritance in vitro inheritance. These tools were also used to demonstrate that Ypt7p must be present on both fusion partners for vacuole fusion.10 Furthermore, we have raised specific antibodies to Sec18p, a yeast protein
(Figure 6A). These antibodies are specific for Ypt7p, as shown by Western blotting (Figure 6B) and control experiments using antibodies raised against other Ypt proteins or purified recombinant Ypt proteins.10 Other ras-like GTPases had previously been implicated in several intracellular fusion events.32,33 The invitro use of specific Ypt7p-antibodies and of components of ypt7∆ cells allowed the identification of this important GTPase as a part of the fusion machinery, the first one identified to play a role in organelle
Figure 5. Microscopic analysis of vacuole fusion reactions. Vacuole fusion reactions contained salt, cytosol and ATP (top left), salt and ATP (top right), salt, cytosol and ATP and affinity-purified Ypt7p-antibodies (middle left), or the same components, but in addition purified recombinant Ypt7p (middle right), or salt, cytosol, ATP and Gdi1p (which extracts Ypt-proteins from membranes; bottom left). The vacuoles were incubated at 25°C for 100 min and visualized by fluorescence microscopy using the vacuole membrane label FM4-64. (Reproduced from Haas et al, The EMBO Journal, 1995, vol. 14, pp. 5258-5270 by copyright permission of Oxford University Press, ref 10.)
Figure 4. Vacuoles are fragmented in ypt7 ∆ cells. Vacuoles were stained in vivo as in Figure 1 (A) or in vitro using the same dye (B), and photographed during fluorescence microscopy. Left panel, YPT7 wild type cells, right panel, isogenic ypt7∆ cells. (Reproduced from Haas et al, The EMBO Journal, 1995, vol. 14, pp. 5258-5270 by copyright permission of Oxford University Press, ref 10).
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Synthesis
with high homology to the mammalian ‘N-ethylmaleimide-sensitive fusion protein’, NSF.34 Sec18p is involved in several steps of intracellular transport in yeast.35,36 Addition of these antibodies to the cell-free vacuole fusion assay, or of antibodies to the Sec18pinteracting protein Sec17p (yeast soluble NSF-attachment protein), inhibited the fusion reactions completely.37 Using control reagents such as monovalent antibodies, preimmune antibodies, and antibodies complexed with their antigens, we could clearly demonstrate that the in-vitro-effect is due to a specific interaction of antibodies. Another advantage of antibodies is that they can be used against the macromolecule in situ under wildtype conditions and do not require the use of components from mutant cells. Of course, this fruitful approach must be controlled for potential indirect effects.
The combined use of morphological, genetic and biochemical methods can now be used to further dissect the inheritance reactions and the components involved. Several new vac mutants (which do not mislocalize vacuolar proteins) have recently been isolated (L. Weisman, personal communication) and will be analysed in the in-vitro assay. We have also purified a novel fusion-stimulating form of thioredoxin using the alkaline phosphatase maturation assay.38 Subsequent analysis of yeast cells with deletions in thioredoxin genes demonstrated that the double deletion mutant has a vac phenotype whereas the single deletion mutants inherited their vacuoles normally.38 Therefore, one functional thioredoxin isoform is necessary for vacuole inheritance in vivo. Similarly, cytosol fractionation has yielded other stimulatory protein fractions (Z. Xu, P. Slusarewicz, A. Haas and W. Wickner, unpublished) which are currently being analysed in detail. We have furthermore identified subreactions which depend on the addition of cytosol, allowing for the purification of additional factors. For example, stage III vacuoles, reisolated after 20 min from an ongoing fusion reaction (standard reaction time, 90 min) and resuspended in only buffer, can fuse in the absence of either cytosol or ATP, requiring only a minimum vacuole concentration and time. Fusion of stage III vacuoles is insensitive to Ypt7p antibodies, indicating that the requirement for Ypt7p-function has largely been fulfilled. However, if cytosol and ATP are incubated together with stage III vacuoles, fusion again becomes sensitive to anti-Ypt7p,10 yielding an assay for the purification of the cytosolic factor which mediates antibody sensitivity. Similarly, fusion of stage III vacuoles is only sensitive to protein phosphatase inhibitors in the presence of both ATP and cytosol,14 suggesting that protein phosphorylation by a cytosolic kinase may inhibit the actual fusion reaction. Such a factor would be particularly interesting with respect to the regulation of organelle inheritance by the cell cycle.39 In general and vacuole inheritance in particular.14 The analysis of the role of non-protein cofactors in vacuole fusion can also best be addressed using the cell-free system. Certain acyl-coenzyme A compounds stimulate vacuole fusion,37 and may do so by acylation of vacuolar proteins. Such an acylation can be followed in vitro.40 We have also recently discovered that the vac5-l mutant22 produces a low molecular weight factor which inhibits vacuole fusion in vitro.29a The versatility of our fusion assay allows for the purifica-
Figure 6. Ypt7p-specific antibodies inhibit homotypic vacuole fusion in vitro. (A) In-vitro fusion reactions contained either affinity-purified antibodies to the GTPase Ypt7p (open squares) or Ypt7p antibodies that had been preincubated with their antigen, Ypt7p (half-filled diamonds). Fusion reactions also contained cytosol, ATP and its regenerating system, and a salt buffer, and were incubated for 120 min at 25°C. Alkaline phosphatase activities were determined and are shown as a function of fusion frequency. (B) Polyclonal antibodies specific for Ypt7p (as used in A) inhibit homotypic vacuole fusion. Immunoblot analysis using affinity-purified anti-Ypt7p. Fifty µg vacuole protein from a YPT7 deletion strain (lane 1), or a YPT7 wild type strain (lane 2) or 100 µg cytosolic protein from a YPT7 wild type strain (lane 3) were separated by electrophoresis, transferred to a membrane, and immunodecorated with an affinity purified Ypt7p-antibody. No immunologically reacting protein can be seen in the deletion strain preparation, Ypt7p and a degradation product thereof are present in the wild type strain preparation, and a small pool of Ypt7p is present in the cytosol. Molecular mass ( 3 1,000) marker proteins are indicated. (Reproduced from Haas et al, The EMBO Journal, 1995, vol. 14, pp. 5258-5270 by copyright permission of Oxford University Press.)
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Vacuole inheritance in vitro 7. Klionsky DJ, Herman PK, Emr SD (1990) The fungal vacuole: composition, function, and biogenesis. Microbiol Rev 54:266-292 8. Raymond CK, Roberts CJ, Moore KE, Howald I, Stevens TH (1992) Biogenesis of the vacuole in Saccharomyces cerevisiae Int Rev Cytol 139:59-120 9. Weisman LS, Bacallao R, Wickner WT (1987) Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J Cell Biol 105:1539-1547 10. Haas A, Scheglmann D, Lazar T, Gallwitz D, Wickner W (1995) The GTPase Ypt7p is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance. EMBO J 14:5258-5270 11. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779-792 12. Weisman LS, Wickner WT (1988) Intervacuole exchange in the yeast zygote: a new pathway in organelle communication. Science 241:289-591 13. Pringle JR, Preston RA, Adam AEM, Stearns T, Drubin DG, Haarer BK, Jones EW (1989) Fluorescence microscopy methods for yeast. Meth Cell Biol 31:357-435 14. Conradt B, Shaw J, Vida T, Emr S, Wickner W (1992) In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol 119:1469-1479 15. Gomes de Mesquita DS, ten Hoopen R, Woldringh CL (1991) Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle. J Gen Microbiol 137:2447-2454 16. Novick P, Fields C, Schekman R (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21:205-215 17. Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936-4948 18. Rothman JH, Howald I, Stevens TH (1989) Characterization of genes required for protein sorting and vacuolar function in the yeast. Saccharomyces cerevisiae. EMBO J 8:2057-2065 19. Banta LM, Robinson JS, Klionsky DJ, Emr SD (1988) Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein assembly. J Cell Biol 107:1369-1383 20. Weisman L, Emr SD, Wickner WT (1990) Mutants of Saccharomyces cerevisiae that block intervacuole vesicular traffic and vacuole division and segregation. Proc Natl Acad Sci USA 87:1076-1080 21. Shaw J, Wickner W (1991) vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting. EMBO J10:1741-1748 22. Nicolson TA, Weisman LS, Payne GS, Wickner WT (1995) A truncated form of the Pho80 cyclin redirects the Pho85 kinase to disrupt vacuole inheritance in S. cerevisiae. J Cell Biol 130:835-845 23. Wada Y, Ohsumi Y, Anraku Y (1992) Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae J Biol Chem 267:18665-18670 24. Weisman L, Emr S, Wickner W (1990) Mutants of S. cerevisiae that block intervacuole traffic and vacuole division and segregation. Proc Natl Acad Sci USA 87:1076-1080 25. Weisman L, Wickner W (1992) Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting. J Biol Chem 267:618-623 26. Jacquemin-Faure I, Thomas D, Laporte J, Cibert C, SurdinKerjan Y (1994) The vacuolar compartment is required for sulfur amino acid homeostasis in Saccharomyces cerevisiae. Mol Gen Genet 244:519-529
tion of such a factor. Further steps will also include the design of new assays for specific subreactions different from vacuole fusion, e.g. assays for the requirements leading to structure formation or the requirements for vacuole docking. As such new assays become available, we will be able to biochemically describe the key players and their interactions, and then use the genetic and morphological analyses of wild-type and mutant cells to challenge the biochemical results and vice versa.
Note added in proof Using our in-vitro fusion reaction, we could recently show that Sec17p and Sec18p are not involved in the lipid bilayer fusion reaction itself, but are required at a very early step, i.e. before or during docking of vacuoles prior to actual fusion. This is in contrast to the leading current hypotheses.34 Mayer A, Wickner W, Haas A (1996) Sec18p (NSF)-driven release of Sec17p (α-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85:83-94.
Acknowledgements We thank (in alphabetical order) B. Conradt, D. Geissert, M. Leonard, A. Mayer, T. Nicolson, J. Shaw, P. Slusarewicz, L. Weisman and Z. Xu for their contributions to the studies presented here, and D. Gallwitz and D. Scheglmann for the successful collaboration. Our studies have been supported by a grant from the National Institutes of General Medical Sciences.
References 1. Birky Jr CW (1983) The partitioning of cytoplasmic organelles at cell division. Int Rev Cytol 15(Suppl):49-89 2. Warren G, Wickner W (1996) Organelle inheritance. Cell 84:395-400 3. Byers B (1981) Cytology of the yeast cell cycle, in Life Cycle and Inheritance (Strathern JN, Jones EW, Broach JR, eds) pp 59-96. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY, USA) 4. Preuss DJ, Mulholland CA, Kaiser CA, Orlean O, Albright CR, Robbins PW, Bostein D (1991) Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy. Yeast 7:891-911 5. Preuss DJ, Mulholland A, Franzusoff A, Segev N, Botstein D (1992) Characterization of the Sacharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol Biol Cell 3:789-803 6. McConnell SJ, Jaffe MP (1993) Intermediate filament formation by a yeast protein essential for organelle inheritance. Science 260:687-689
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A. Haas and W. Wickner 27. Pryer NK, Wuestehube LJ, Schekman R (1992) Vesicle-mediated protein sorting. Annu Rev Biochem 61:471-516 28. Baker D, Hicke L, Rexach M, Schleyer M, Schekman R (1988) Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell 54:335-344 29. Haas A, Conradt B, Wickner W (1994) G-protein ligands inhibit in vitro reactions of vacuole inheritance. J Cell Biol 126:87-97 29a. Nicolson T, Conradt B, Wickner W (1996) A truncated form of the Pho80 cyclin of Saccharomyces cerevisiae induces expression of a small aytosolic factor which inhibits vacuole inheritance. J Bacteriol, in press 30. Conradt B, Haas A, Wickner W (1994) Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro. J Cell Biol 126:99-110 31. Wichmann H, Hengst L, Gallwitz D (1992) Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p). Cell 71:1131-1142 32. Ferro-Novick S, Novick P (1993) The role of GTP-binding proteins in transport along the exocytic pathway. Annu Rev Cell Biol 9:575-599
33. Nuoffer C, Balch WE (1994) GTPases: multifunctional switches regulating vesicular traffic. Annu Rev Biochem 63:949-990 34. Rothman JE (1994) Mechanisms of intracellular membrane fusion. Nature 372:55-63 35. Eakle KA, Bernstein M, Emr SD (1988) Characterization of a component of the yeast secretory machinery: Identification of the SEC18 gene product. Mol Cell Biol 8:4098-4109 36. Graham TR, Emr SD (1991) Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol 114:207-218 37. Haas A, Wickner W (1996) Homotypic vacuole fusion requires Sec17p (yeast α-SNAP) and Sec18p (yeast NSF). EMBO J 15:3296-3305 38. Xu Z, Wickner W (1996) Thioredoxin is required for vacuole inheritance in S. cerevisiae. J Cell Biol 132:787-794 39. Nunnari J, Walter P (1996) Regulation of organelle biogenesis. Cell 84:389-394 40. Mundy DI (1995) Protein palmitoylation in membrane trafficking. Biochem Soc Transacts 116:135-146
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CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 517–524
Organelle inheritance in a test tube: the yeast vacuole Albert Haas and William Wickner
these organelles in vivo and in vitro;7,9-13 (iii) several marker proteins for vacuoles have been characterized and their genes cloned,7 making vacuole analysis easier; (iv) powerful genetic tools are available for the study of S. cerevisiae and it can be readily grown in large quantities using simple methods.
Vacuoles (lysosomes) of the budding yeast Saccharomyces cerevisiae are low copy-number organelles which are inherited from mother to daughter cells. We are interested in the molecular basis of the division and segregation of vacuole material and in the coordination of these events with the cell cycle. Using microscopic, genetic and biochemical methods, we have shown that vacuole inheritance is a regulated process, described several genes and gene products involved in the course of inheritance, and developed a cell-free system which allows an in-vitro reconstitution of events that occur during vacuole inheritance in vivo.
The classical morphological approach: vacuole inheritance in vivo Initial microscopic studies showed that the growing bud receives most of its vacuolar material from the mother cell.9 Further analysis, using yeast zygotes, demonstrated that there is a ‘communication’ of vacuolar material between the two mating partners.12 Certain ‘tracks’ clearly exist in a zygote which conduct vesicles derived from either parental cell vacuole. These vesicles establish the vacuole in the emerging bud. At the same time, these tracks always run from parental vacuoles into the bud, never directly between the vacuoles from the mating partner (a- and α-) cells.12 Further studies showed that specific tracks are also used for the transport of vacuolar vesicles when haploid yeast cells were examined.14,15 Obviously, vesicular and tubular ‘segregation structure’ formation from the mother vacuole is coordinated by the cell cycle (occurring between S phase and G2/M phase transition) and segregation structure transport into the bud is spatially restricted to these direct connections (Figure 1). As the last discernible step of vacuole inheritance, the segregation structures fuse, allowing both mother and separated daughter cells to possess one or a few vacuoles (Figure 1; ref 14).
Key words: membrane fusion / organelle inheritance / vacuole / yeast ©1996 Academic Press Ltd
CYTOPLASMIC ORGANELLES are not produced de novo after cell division but arise from the growth, multiplication and separation of preexisting organelles. High copy-number particles, such as ribosomes, may be inherited by random diffusion. Inheritance of low copy-number organelles, however, requires a cell cycle-regulated multiplication and separation processes to ensure a proper partitioning between mother and daughter cells.1,2 In the budding yeast Saccharomyces cerevisiae, the inheritance of the nucleus,3 endoplasmic reticulum,4 Golgi apparatus,5 and mitochondria6 have been analysed. However, we are only at the start of studies of the molecular mechanisms underlying organelle multiplication and partitioning. This laboratory is interested in the analysis of the molecular mechanisms which enable baker’s yeast to faithfully inherit vacuoles7,8 during cell multiplication. We have chosen this organism and this organelle for practical reasons: (i) Vacuoles are very large lowcopy organelles and therefore can be observed by phase-contrast optics;9 (ii) specific fluorophores are available which accumulate in the acidic, esterase-rich vacuolar lumen and can be used to readily identify
The genetic approach: temperature-sensitive inheritance mutants The analysis of temperature-sensitive yeast mutants in secretion (sec mutants) or in vacuole protein sorting (vps mutants) have profoundly influenced our understanding of these pathways.7,16-18 The temperaturesensitive phenotype allows for the isolation of the respective genes by complementation of the mutated
From the Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3840, USA ©1996 Academic Press Ltd 1084-9521/96/040517 + 08 $18.00/0
517
A. Haas and W. Wickner sensitivity of the strains to calcium, lysine, histidine or calcium, defects in sulfur metabolism, or screening for vacuole protease defects. The same gene (VP33) can therefore have several aliases, here CLS14, MET27, PEP14, SLP1, VPT33 and VAM5 (see ref 26).
locus. Analysis of mutants for a defect in vacuole morphology, however, is more difficult because of the lack of a phenotype which controls cell growth. Only about 50% of all mutants without a vacuole are temperature-sensitive.19,20 Therefore, the tedious microscopic screening of temperature-sensitive mutants,21 or a screen based on the somewhat higher density of cells lacking vacuoles,22 or survival in the presence of chloroquine23 were used to isolate vacuole inheritance (vac) mutants. Also, pink (or white) derivatives of otherwise red ade-2 yeast colonies showed an increased incidence of vac mutations providing a colour screening procedure.23 The analysis of vac mutants has yielded important insights. It has been established that vacuole inheritance and vacuole protein sorting can be studied separately. Some vac mutants, like vac1-1 ( = vps19),24,25 missort proteins which are normally delivered to the vacuole and, at the same time, fail to deliver vacuoles to the bud at non-permissive temperatures. The vac2-1 mutant, however, has a vacuole inheritance defect but no protein sorting defect.21 Interestingly, some vacuole morphology mutants, such as vps33, were identified by several independent screens, such as vacuole protein missorting, hyper-
The in vitro biochemical approach: taking apart the subreactions This approach is based on the classic reductionist approach of enzymology. It assumes that complex biochemical reactions which occur in the cell can also occur in a cell-free environment under the proper conditions. Molecular analysis then becomes feasible, as the environment can be shaped by adding or omitting isolated factors. In fact, there are several examples of successful reconstitutions of intracellular trafficking reactions.27 As a first step to a biochemical dissection of vacuole inheritance processes, we have employed semi-intact yeast cells.14 These are spheroplasts that have been permeabilized through a combination of osmotic shock and slow freezing.28 They retain much of their organellar architecture, yet are permeable to low-
Figure 1. Vacuole morphology during the cell cycle of S. cerevisiae. The top portion shows a schematic representation of the events recorded in the photographs in the lower portion. Yeast cells were labeled in vivo using the fluorogenic, vacuole-staining dye CDCFDA and observed by fluorescence microscopy. The phases of the cell cycle represented by the cells shown are indicated. (Reproduced from Conradt et al, The Journal of Cell Biology, 1992, vol. 119, pp. 1469-1479 by copyright permission of The Rockefeller University Press, ref 14.)
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Vacuole inheritance in vitro ‘fusion’ reactions we are observing require ‘upstream’ components of the inheritance apparatus (e.g. priming or vesiculation factors). Our cell-free system requires proteins which are sensitive to pharmacological reagents such as mastoparan (a G-protein activator), GTPγS (which is only very slowly hydrolysed by GTPases), neomycin (which binds strongly to polyphosphoinositides), bafilomycin (an inhibitor of vacuolar proton-pumping ATPases), and microcystin-LR and okadaic acid (inhibitors of serine/threonine protein phosphatases). Using these inhibitors,14,29,30 we could show that the overall reaction in vitro actually consists of several steps. Four subreactions have been defined: first, the isolated vacuoles must be briefly in contact with physiological salt concentrations; second, they must be incubated with cytosolic components; third, vacuoles react with added ATP, and fourth, the final stages of the reaction require incubation of vacuoles at a minimal vacuole concentration and a physiological temperature. The fusion reaction requires these steps in precisely this sequence.30 Although the question remains whether
molecular weight substances and to macromolecules, such as soluble proteins. Segregation structure formation in such cells, which lack much of their soluble components, was dependent on the external addition of energy (ATP) and was stimulated by the addition of a preparation of cytosol. These reactions did not occur, however, when vac1 or vac2 cells and cell components were used in the experiments, underscoring the authenticity of the reactions observed.14 These data suggested that reconstitution of vacuole inheritance reactions in a cell-free system should, in principle, be feasible. In the next step, we have therefore analysed vacuole information reactions using isolated components. Isolated vacuoles not only form segregation-like structures, but also fuse in the test tube when they are incubated with cytosol, an ATP-regenerating system and a physiological salt mixture at a physiological temperature (25 to 30°C).14 However, when components of the in-vitro reactions were isolated from vac mutants, the vacuoles fragmented and/or would not fuse.10,14,29 This close correlation between in-vivo and in-vitro observations allowed us to conclude that the reactions seen in the test tube are authentic reconstitutions of reactions occuring in the living cell. Encouraged by this reconstitution of at least two steps of vacuole inheritance (structure formation and vacuole fusion) in a cell-free system, we established a modified in-vitro assay which would allow us to quantify fusion frequency biochemically rather than by microscopic observation of vacuole morphology and size (Figure 2). The basis for this system is the observation that vacuoles possess a membrane-bound alkaline phosphatase (Pho8p) which reaches into the vacuolar lumen and which is imported as an enzymatically inactive precursor (proPho8p). In the vacuole lumen, proPho8p is cleaved by the vacuolar proteinase A (Pep4p), and thus gains alkaline phosphatase activity. Therefore, the vacuoles used in the standard cell-free reaction are isolated from two yeast strains, one with a deletion in the PHO8 gene, the other with a deletion in the PEP4 gene. Neither vacuole population has alkaline phosphatase activity. Enzymatic activity can only be generated by the mixing of the luminal contents of both vacuole types, and hence is a measure of frequency of vacuole fusion in vitro (Figure 2 and 3; ref 29). This in-vitro vacuole fusion reaction is again defective when components are used from vac-1, vac-2 or vac-5 cells (refs 10,14,29,29a), and therefore represent physiologically authentic reactions. Also, studying components from several vac cells in our cell-free reactions, it became clear that the
Figure 2. Scheme describing the pro-alkaline phosphatase maturation assay which is routinely used to quantitate homotypic vacuole fusion. For details see text. (Reproduced from Haas et al, The Journal of Cell Biology, 1994, vol. 126, pp. 87-97 by copyright permission of The Rockefeller University Press, ref 29.)
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A. Haas and W. Wickner the identical events occur in intact yeast cells, this study nevertheless allows for the isolation of stagespecific factors. One of the particular advantages of a cell-free system is the possibility of using specific antibodies to probe for the involvement of certain factors in the
reaction. Yeast cells mutated in the ras-like GTPase Ypt7p have fragmented vacuoles31 (Figure 4). Using specific Ypt7p-antibodies, we could show that this protein is absolutely required for homotypic (self-) vacuole fusion in both, the microscopic fusion assay (Figure 5) and the pro-Pho8p maturation assay
Figure 3. Kinetics of cell-free vacuole-to-vacuoles fusion. All parts of this figure are from the same experiment. (A) A mixture of vacuoles from pho8 and pep4 strains was incubated with cytosol, an ATP-regenerating system and salt at 25°C. Vacuoles fuse over time, as seen by a decrease in vacuole number and an increase in their average size. The vacuoles have been stained with a vacuolespecific fluorphor. (B) In parallel, the maturation of proPho8p was analysed by immunoblotting reaction samples removed at various times. (C) At various times, samples were removed and their alkaline phosphatase activities determined. These parallel experiments clearly demonstrate that generation of alkaline phosphatase activity parallels the enlargement of vacuoles and therefore represents homotypic vacuole fusion. (Reproduced from Haas et al, The Journal of Cell Biology, 1994, vol. 126, pp. 87-97 by copyright permission of The Rockefeller University Press, ref 29.)
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Vacuole inheritance in vitro inheritance. These tools were also used to demonstrate that Ypt7p must be present on both fusion partners for vacuole fusion.10 Furthermore, we have raised specific antibodies to Sec18p, a yeast protein
(Figure 6A). These antibodies are specific for Ypt7p, as shown by Western blotting (Figure 6B) and control experiments using antibodies raised against other Ypt proteins or purified recombinant Ypt proteins.10 Other ras-like GTPases had previously been implicated in several intracellular fusion events.32,33 The invitro use of specific Ypt7p-antibodies and of components of ypt7∆ cells allowed the identification of this important GTPase as a part of the fusion machinery, the first one identified to play a role in organelle
Figure 5. Microscopic analysis of vacuole fusion reactions. Vacuole fusion reactions contained salt, cytosol and ATP (top left), salt and ATP (top right), salt, cytosol and ATP and affinity-purified Ypt7p-antibodies (middle left), or the same components, but in addition purified recombinant Ypt7p (middle right), or salt, cytosol, ATP and Gdi1p (which extracts Ypt-proteins from membranes; bottom left). The vacuoles were incubated at 25°C for 100 min and visualized by fluorescence microscopy using the vacuole membrane label FM4-64. (Reproduced from Haas et al, The EMBO Journal, 1995, vol. 14, pp. 5258-5270 by copyright permission of Oxford University Press, ref 10.)
Figure 4. Vacuoles are fragmented in ypt7 ∆ cells. Vacuoles were stained in vivo as in Figure 1 (A) or in vitro using the same dye (B), and photographed during fluorescence microscopy. Left panel, YPT7 wild type cells, right panel, isogenic ypt7∆ cells. (Reproduced from Haas et al, The EMBO Journal, 1995, vol. 14, pp. 5258-5270 by copyright permission of Oxford University Press, ref 10).
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Synthesis
with high homology to the mammalian ‘N-ethylmaleimide-sensitive fusion protein’, NSF.34 Sec18p is involved in several steps of intracellular transport in yeast.35,36 Addition of these antibodies to the cell-free vacuole fusion assay, or of antibodies to the Sec18pinteracting protein Sec17p (yeast soluble NSF-attachment protein), inhibited the fusion reactions completely.37 Using control reagents such as monovalent antibodies, preimmune antibodies, and antibodies complexed with their antigens, we could clearly demonstrate that the in-vitro-effect is due to a specific interaction of antibodies. Another advantage of antibodies is that they can be used against the macromolecule in situ under wildtype conditions and do not require the use of components from mutant cells. Of course, this fruitful approach must be controlled for potential indirect effects.
The combined use of morphological, genetic and biochemical methods can now be used to further dissect the inheritance reactions and the components involved. Several new vac mutants (which do not mislocalize vacuolar proteins) have recently been isolated (L. Weisman, personal communication) and will be analysed in the in-vitro assay. We have also purified a novel fusion-stimulating form of thioredoxin using the alkaline phosphatase maturation assay.38 Subsequent analysis of yeast cells with deletions in thioredoxin genes demonstrated that the double deletion mutant has a vac phenotype whereas the single deletion mutants inherited their vacuoles normally.38 Therefore, one functional thioredoxin isoform is necessary for vacuole inheritance in vivo. Similarly, cytosol fractionation has yielded other stimulatory protein fractions (Z. Xu, P. Slusarewicz, A. Haas and W. Wickner, unpublished) which are currently being analysed in detail. We have furthermore identified subreactions which depend on the addition of cytosol, allowing for the purification of additional factors. For example, stage III vacuoles, reisolated after 20 min from an ongoing fusion reaction (standard reaction time, 90 min) and resuspended in only buffer, can fuse in the absence of either cytosol or ATP, requiring only a minimum vacuole concentration and time. Fusion of stage III vacuoles is insensitive to Ypt7p antibodies, indicating that the requirement for Ypt7p-function has largely been fulfilled. However, if cytosol and ATP are incubated together with stage III vacuoles, fusion again becomes sensitive to anti-Ypt7p,10 yielding an assay for the purification of the cytosolic factor which mediates antibody sensitivity. Similarly, fusion of stage III vacuoles is only sensitive to protein phosphatase inhibitors in the presence of both ATP and cytosol,14 suggesting that protein phosphorylation by a cytosolic kinase may inhibit the actual fusion reaction. Such a factor would be particularly interesting with respect to the regulation of organelle inheritance by the cell cycle.39 In general and vacuole inheritance in particular.14 The analysis of the role of non-protein cofactors in vacuole fusion can also best be addressed using the cell-free system. Certain acyl-coenzyme A compounds stimulate vacuole fusion,37 and may do so by acylation of vacuolar proteins. Such an acylation can be followed in vitro.40 We have also recently discovered that the vac5-l mutant22 produces a low molecular weight factor which inhibits vacuole fusion in vitro.29a The versatility of our fusion assay allows for the purifica-
Figure 6. Ypt7p-specific antibodies inhibit homotypic vacuole fusion in vitro. (A) In-vitro fusion reactions contained either affinity-purified antibodies to the GTPase Ypt7p (open squares) or Ypt7p antibodies that had been preincubated with their antigen, Ypt7p (half-filled diamonds). Fusion reactions also contained cytosol, ATP and its regenerating system, and a salt buffer, and were incubated for 120 min at 25°C. Alkaline phosphatase activities were determined and are shown as a function of fusion frequency. (B) Polyclonal antibodies specific for Ypt7p (as used in A) inhibit homotypic vacuole fusion. Immunoblot analysis using affinity-purified anti-Ypt7p. Fifty µg vacuole protein from a YPT7 deletion strain (lane 1), or a YPT7 wild type strain (lane 2) or 100 µg cytosolic protein from a YPT7 wild type strain (lane 3) were separated by electrophoresis, transferred to a membrane, and immunodecorated with an affinity purified Ypt7p-antibody. No immunologically reacting protein can be seen in the deletion strain preparation, Ypt7p and a degradation product thereof are present in the wild type strain preparation, and a small pool of Ypt7p is present in the cytosol. Molecular mass ( 3 1,000) marker proteins are indicated. (Reproduced from Haas et al, The EMBO Journal, 1995, vol. 14, pp. 5258-5270 by copyright permission of Oxford University Press.)
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Vacuole inheritance in vitro 7. Klionsky DJ, Herman PK, Emr SD (1990) The fungal vacuole: composition, function, and biogenesis. Microbiol Rev 54:266-292 8. Raymond CK, Roberts CJ, Moore KE, Howald I, Stevens TH (1992) Biogenesis of the vacuole in Saccharomyces cerevisiae Int Rev Cytol 139:59-120 9. Weisman LS, Bacallao R, Wickner WT (1987) Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J Cell Biol 105:1539-1547 10. Haas A, Scheglmann D, Lazar T, Gallwitz D, Wickner W (1995) The GTPase Ypt7p is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance. EMBO J 14:5258-5270 11. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779-792 12. Weisman LS, Wickner WT (1988) Intervacuole exchange in the yeast zygote: a new pathway in organelle communication. Science 241:289-591 13. Pringle JR, Preston RA, Adam AEM, Stearns T, Drubin DG, Haarer BK, Jones EW (1989) Fluorescence microscopy methods for yeast. Meth Cell Biol 31:357-435 14. Conradt B, Shaw J, Vida T, Emr S, Wickner W (1992) In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol 119:1469-1479 15. Gomes de Mesquita DS, ten Hoopen R, Woldringh CL (1991) Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle. J Gen Microbiol 137:2447-2454 16. Novick P, Fields C, Schekman R (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21:205-215 17. Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936-4948 18. Rothman JH, Howald I, Stevens TH (1989) Characterization of genes required for protein sorting and vacuolar function in the yeast. Saccharomyces cerevisiae. EMBO J 8:2057-2065 19. Banta LM, Robinson JS, Klionsky DJ, Emr SD (1988) Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein assembly. J Cell Biol 107:1369-1383 20. Weisman L, Emr SD, Wickner WT (1990) Mutants of Saccharomyces cerevisiae that block intervacuole vesicular traffic and vacuole division and segregation. Proc Natl Acad Sci USA 87:1076-1080 21. Shaw J, Wickner W (1991) vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting. EMBO J10:1741-1748 22. Nicolson TA, Weisman LS, Payne GS, Wickner WT (1995) A truncated form of the Pho80 cyclin redirects the Pho85 kinase to disrupt vacuole inheritance in S. cerevisiae. J Cell Biol 130:835-845 23. Wada Y, Ohsumi Y, Anraku Y (1992) Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae J Biol Chem 267:18665-18670 24. Weisman L, Emr S, Wickner W (1990) Mutants of S. cerevisiae that block intervacuole traffic and vacuole division and segregation. Proc Natl Acad Sci USA 87:1076-1080 25. Weisman L, Wickner W (1992) Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting. J Biol Chem 267:618-623 26. Jacquemin-Faure I, Thomas D, Laporte J, Cibert C, SurdinKerjan Y (1994) The vacuolar compartment is required for sulfur amino acid homeostasis in Saccharomyces cerevisiae. Mol Gen Genet 244:519-529
tion of such a factor. Further steps will also include the design of new assays for specific subreactions different from vacuole fusion, e.g. assays for the requirements leading to structure formation or the requirements for vacuole docking. As such new assays become available, we will be able to biochemically describe the key players and their interactions, and then use the genetic and morphological analyses of wild-type and mutant cells to challenge the biochemical results and vice versa.
Note added in proof Using our in-vitro fusion reaction, we could recently show that Sec17p and Sec18p are not involved in the lipid bilayer fusion reaction itself, but are required at a very early step, i.e. before or during docking of vacuoles prior to actual fusion. This is in contrast to the leading current hypotheses.34 Mayer A, Wickner W, Haas A (1996) Sec18p (NSF)-driven release of Sec17p (α-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85:83-94.
Acknowledgements We thank (in alphabetical order) B. Conradt, D. Geissert, M. Leonard, A. Mayer, T. Nicolson, J. Shaw, P. Slusarewicz, L. Weisman and Z. Xu for their contributions to the studies presented here, and D. Gallwitz and D. Scheglmann for the successful collaboration. Our studies have been supported by a grant from the National Institutes of General Medical Sciences.
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