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[ 4 4 ] M e t h o d s for S t u d y i n g t h e Y e a s t V a c u o l e

By CHRISTOPHER J.

ROBERTS, CHRISTOPHER K. RAYMOND, CARL T. YAMASHIRO, and TOM H. STEVENS

Introduction The yeast vacuole is the equivalent oflysosomes in animal cells and the vacuoles of plant cells. It is an acidic compartment that contains a large number of hydrolases. 1-3 Use of the genetic techniques available in yeast, together with biochemical and cell biological approaches, has shed light on many aspects of the vacuole, including (1) the structure and biosynthesis of several vacuolar proteins, 1,4-6 (2) the localization determinants on vacuolar proteins that are necessary and sufficient for proper targeting to the vacuole, 7-9 (3) the identification of genes necessary for vacuolar protein sorting aT,d organelle biogenesis (TIPS genes, vacuolar t~rotein _sorting)1°-15 and acidification of the lumen of the vacuole, 13,m6.~7(4) the composition and biochemical characteristics of the vacuolar H+-ATPase known to be necessary for the generation of the acidic interior of the vacuole, 18-2° and (5) the behavior of the vacuole during the cell cycle. 2~-ub

J. H. Rothman and T. H. Stevens, in "Protein Transfer and Organelle Biogenesis" (R. Das and P. Robbins, eds.), p. 159. Academic Press, San Diego, 1988. 2 D. J. Klionsky, L. M. Banta, J. S. Robinson, and S. D. Emr, in "Molecular Biology of Intracellular Protein Sorting and Organelle Assembly" (R. Bradshaw, L. McAllister-Henn, and M. Douglas, eds.), p. 173. Alan R. Liss, New York, 1988. 3 j. H. Rothman, C. T. Yamashiro, P. M. Kane, and T. H. Stevens, Trends Biochem. Sci. 14, 347 (1989). 4 B. Mechler, H. H. Hirsch, H. Mfiller, and D. H. Woff, EMBOJ. 7, 1705 (1988). 5 C. M. Moehle, C. K. Dixon, and E. W. Jones, J. Cell Biol. 108, 309 (1989). C. J. Roberts, G. Pohtig, J. H. Rothman, and T. H. Stevens, J. CellBiol. 108, 1363 (1989). L. A. Vails, C. P. Hunter, J. H. Rothman, and T. H. Stevens, Cell (Cambridge, Mass.) 48, 887 (1987). 8 L. M. Johnson, V. A. Bankaitis, and S. D. Emr, Cell (Cambridge, Mass.) 48, 875 (1987). 9 D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8, 2105 (1988). l0 j. H. Rothman and T. H. Stevens, Cell (Cambridge, Mass.) 47, 1041 (1986). H V. A. Bankaitis, L. M. Johnson, and S. D. Emr, Proc. Natl. Acad. ScL U.S.A. 83, 9075 (1986). 12j. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Moi. Ceil. Biol. 8, 4936 (1988). ~3L. M. Banta, J. S. Robinson, D. J. Klionsky, and S. D. Emr, J. CellBiol. 107, 1369 (1988). t4 j. H. Rothman, I. Howald, and T. H. Stevens, EMBO J. 8, 2957 (1989). ~ V. Dulic and H. Riezman, EMBO J. 8, 1349 (1989). t6 j. H. Rothman, C. T. Yamashiro, C. IC Raymond, P. M. Kane, and T. H. Stevens, J. Cell Biol. 109, 93 (1989).

METHODSIN ENZYMOLOGY,VOL. 194

Copyright©1991byAc~,micPrem,Inc.

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The purpose of this chapter is to describe techniques used in the study of the vacuole. We focus attention on the procedures used in our laboratory, and, where possible, we try to describe (or at least reference) the methods used by others. The chapter is divided into three sections. In the first section, methods for visualizing vacuoles by light and fluorescence microscopy are outlined. The next section describes a method for purification of vacuoles, marker enzyme assays used to assess yield and purity, and activities of the purified organdie that have been described. The final section describes two methods used in our laboratory for assessing the fidelity of protein sorting to the vacuole. Unless otherwise noted, all reagents used in these procedures are available from Sigma Chemical Co. (St. Louis, MO). Visualization of Yeast Vacuoles by Fluorescence Microscopy

General Remarks The yeast vacuole is a large, dynamic organelle which can be detected by fight microscopy. It is most prominent when cells are viewed using differential interference-contrast optics (commonly referred to as Nomarski optics). However, the vacuoles of many cells in a wild-type population are not readily visualized by Nomarski optics. In this section, we outline procedures for more reliable detection of vacuoles using vital staining with fluorescent dyes in conjunction with fluorescence microscopy. In addition, we discuss immunofluorescence techniques for observation of vacuolar antigens in fixed cells. Two important issues regarding the visualization of yeast vacuoles are the specificity of the stains used and the morphology of vacuoles observed under various conditions. The vital stains discussed below have been shown to specifically label the region which coincides with the vacuole as t7 R. A. Preston, R. F. Murphy, and E. W. Jones, Proc. Natl. Acad. Sci. U.S.A. 86, 7027 (1989). ts E. Uchida, Y. Ohsumi, and Y. Anraku, J. Biol. Chem. 260, 1090 (1985). ~9p. M. Kane, C. T. Yamashiro, J. H. Rothman, and T. H. Stevens, J. Cell Sci. Suppl. 11, 161 (1989). 20p. M. Kane, C. T. Yamashiro, and T. H. Stevens, J. Biol. Chem. 264, 19236 (1989). 21A. Wiemken, P. Matile, and H. Moor, Arch. Mikrobiol. 70, 89 (1970). 22L. H. Hartwell, Proc. Nati. Acad. Sci. U.S.A. 66, 352 (1970). 23L. S. Weisman, R. P~e~dlao, and W. Wickner. J. CellBiol. 105, 1539 (1987). 24, L. S. Weisman and W. Wickner, Science 241, 589 (1988). Ub C. IC Raymond, P. J. O'I-Iara, G. Eichinger, J. H. Rothman, and T. H. Stevens, J. Cell Biol., in press, (1989).

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seen by Nomarski opticS. 23'25'26 These same stains fail to label particular mutants of yeast which are defective in the assembly of the vacuole? 3,~5~ With respect to immunofluorescence applications on fixed cells, it is essential to demonstrate that the antibodies used are specific for the vacuolar protein of interest. The morphology of the vacuole is highly variable and sensitive to many factors, including growth conditions and the genetic background of a particular strain. The vacuole is visible at all stages of the cell cycle and becomes apparent in the bud soon after emergence.23,24bUnder conditions of rapid growth, most cells of the yeast strains commonly used in our laboratory exhibit several small vacuolar compartments which assume a variety of shapes and sizes. Other conditions, such as transfer to glucosefree buffers or approach to stationary phase, appear to cause these smaller vacuoles to coalesce into a large, roughly spherical vacuole. What constitutes normal vacuolar morphology has not been resolved.23,24b,25 This section is limited to the application of fluorescence microscopy to the study of yeast vacuole. For a comprehensive overview of fluorescence microscopy application in yeast cell biology, see Refs. 25 and 27.

Vital Staining A variety of fluorescent probes have been used to label vacuoles in living yeast cells. 25 Among these are membrane-impermeant compounds, including the suffonate dyes (e.g., Lucifer YeUow26.2s) and fluorescein derivatives, ~3,~Ta3a9lysosomotropic dyes, such as quinacrine, ~3't6a3 and endogenously produced fluorophores that accumulate in the vacuoles of several yeast mutants, such as in ade2 m u t a n t s . 13,23 A l t h o u g h all of the molecules specifically label vacuoles, little is known about the mechanisms by which they are concentrated in these organelles. Three protocols for vital staining of yeast vacuoles are given below. The first and second utilize the fluorescein derivatives 5- (and 6-)carboxy-2',7'dichiorofluorescein diacetate (CDCFDA; Molecular Probes, Eugene, OR; Cat. No. C-369) and 6-carboxyfluorescein diacetate (CFDA; Molecular Probes, Inc., Eugene, OR; Cat. No. C-195), which are relatively easy to use and which stain the vacuoles of many different strains reliably (Fig. 1A,B)? 7a5 The third protocol describes the use of quinacrine, a weakly 25j. R. Pringle, R. A. Preston, A. E. M. Adams, T. Stearns, D. G. Drubin, B. IL Haarer, and E. W. Jones, Methods CellBiol. 31, 357 (1989). 26H. P.Jezman, Cell (Cambridge, Mass.) 40, 1001 (1985). 2~j. R. Pringle, this volume [52]. 2s V. Dulic, M. Egerton, L Elguindi, S. Ratl~ B. Singer, and H. Riezman, this volume [48]. 29 R. A. Preston, R. F. Murphy, and E. W. Jones, J. CellBiol. 105, 1981 (1987).

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basic dye which accumulates in low-pH compartments, such as the vacuole.23 Quinacrine accumulation has been used to screen the vps mutants for those which are defective in acidification of the vacuole. 13,meHowever, the amount of dye taken up in different yeast strains can vary, and thus quinacrine is a less reliable vital stain than CDCFDA or CFDA.

Solutions for Vital Staining of Yeast Cells CDCFDA (1000×): 10 mM, prepared in dimethylformamide; stable for months at - 20 ° CFDA (1000×): 10 mM, prepared in dimethylformamide; stable for months at - 20 ° 20 × Phosphate-buffered saline (PBS): Dissolve 160 g NaC1, 22.8 g Na2HPO4, and 4 g KH2PO4 in 1 liter of water; adjust the pH to 7.3 with NaOH Phosphate-buffered YEPD: YEPD buffered with 50 m M Na2HPO4, pH 7.6 Staining with CDCFDA. Suspend approximately 1 × 107 ceils/ml fi'om a fresh plate or actively growing liquid culture in SD minimal broth supplemented with the appropriate nutrients plus 50 mM citric acid, pH 5.0, and 5-10 #M CDCFDA. Incubate with shaking at 30 ° for 1030 rain. Spot 8/zl on a concanavalin A (Con A)-coated microscope slide (prepare Con A-coated slides by spreading 10/d of 1 mg/ml Con A onto a slide and allowing to air dry; the Con A coating causes the cells to adhere to the slide). Cover the cells with a coverslip and view immediately. The CDCFDA-derived fluorescence can be viewed with standard fluorescein optics (Fig. 1A,B). Staining with CFDA. Staining cells with CFDA is performed as with CDCFDA, except the cells are incubated in SD plus nutrients, 50 mM citrate, pH 3.0, and 5 - 1 0 / ~ / C F D A , and the incubation should be extended to 30 min at 30 ° (Fig. IC, D). Notes. Staining may also be performed in buffered YEPD broth, but cells will not stick to Con A-coated slides. The cells will adhere if they are washed with PBS plus 2% glucose after labeling. In our hands and as reported by others,~ the composition of the wash buffer greatly influences vacuolar morphology. Washing with buffers containing glucose yields many cells with several vacuoles, whereas cells washed in solutions free of glucose predominantly possess a single large vacuole (Fig. 1). The fluorescent compounds which accumulate in yeast vacuoles during CDCFDA and CFDA staining are most likely carboxy-2',7'-dichlorofluorescein and carboxyfluorescein (CF), respectively, or a polymerization product of these compoundS.25 These compounds photobleach rapidly and also leak out of the vacuole with a t~/2 of about 30 min at 300; therefore, it

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is necessary to view the cells immediately after staining,. CF undergoes pH-dependent changes in both the absorption and fluorescence emission spectra. This property has been exploited to determine vacuolar pH and to screen for mutants defective in maintenance of a low vacuolar pH. ~7 CDCFDA and CFDA have similar staining properties, but CDCFDA can be used over a wider range of pH with shorter labeling periods, and the fluorescence signal from this compound is brighter than CFDA. Staining with Quinacrine. Harvest ~5 X 106 log-phase yeast cells, resuspend in 500/zl of phosphate-buffered YEPD containing 200/zM quinacrine in a microfuge tube, and incubate for 5 min at room temperature. Harvest the cells by a 5-sec centrifngation, and wash once in 500/d of minimal media or 2% glucose, either of which should be buffered to pH 7.6 with 50 m M Na2HPO4. Resuspend in 100/d of the same solution and apply 10/zl to a Con A-coated microscope slide. Quinacrine fluorescence can be viewed using standard fluorescein fluorescence wavelengths. Notes. Quinacrine-labeled cells may be viewed up to 15 rain after labeling. As with the CDCFDA-labeling protocol, the entire procedure can be carried out in phosphate-buffered YEPD if immobilizing the cells to slides is not critical.

Immunofluorescence Immunofluorescence techniques have been applied successfully to the study of the yeast vacuole (Fig. IE-H). 6,9 In this section we outline the procedures used in our laboratory, with emphasis placed on the techniques that have been most critical for success, including increasing the specificity of antibodies, treatment of fixed cells with sodium dodecyl sulfate (SDS), and enhancement of weak signals by antibody amplification. We typically use rabbit polyclonal antibodies directed against yeast antigens that have been expressed in and purified from Escherichia coll. Subsequent affinity purification of these antibodies has been essential for immunofluorescence experiments. Affinity columns are prepared by at-

FIG. 1. Fluorescence detection of the yeast vacuole in living and fixed cells. (A) and (B) show vacuoles in living cells stained with CDCFDA and applied directly to slides without washing. (C) and (D) show cells stained with CFDA and washed in glucose-free buffer. Indirect immunofluorescence microscopy of fixed yeast cells is shown in (E)-(H). Fixed cells were labeled with either rabbit anti-alkaline phosphatase antibody (E, F) or anti-CPY antibody (G, H) followed by fluorescein-conjugated goat anti-rabbit antibody. Alkaline phosphatase is a vacuolar membrane protein, and CPY is a soluble vacuolar protein. Cells were photographed through Nomarski optics (A, C, E, and G) or by fluorescein fluorescence (B, D, F, and H).

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taching the original immunogen to CNBr-activated Sepharose beads, and affinity purification of antibodies is carried out essentially as described. 25,3°,3~We have found that two successive purifications of the antibodies (double-affinity purification) results in optimal antibody titer and specificity. Successful antigen detection in immunofluorescence experiments is best monitored by comparison of the staining pattern observed in wildtype cells to that of a deletion strain, that is, a strain in which the gene encoding the protein of interest has been deleted, if this is possible. If a background signal is seen in the deletion strain then further purification of the antibody is necessary. We have accomplished this by adsorbing small amounts of afffinity-purified antibodies to deletion strain cells that have been fixed and prepared as described below. Apparently this procedure improves the signal-to-noise ratio by removing nonspecific antibodies. We use well-established techniques for indirect immunofluorescence detection of proteins in yeast. 25.32A protocol for treatment of spheroplasted fixed cells with antibodies on polylysine-coated slides is described below. Also, we describe a signal amplification procedure that has been used successfully for immunolocalization of nonabundant antigens. This protocol has worked well using polyclonal and monoclonal antibodies for staining of both soluble and membrane-associated vacuolar antigens. These include dipeptidyl aminopeptidase B (DPAP B),6 carboxypeptidase Y (CPY), alkaline phosphatase, proteinase B (PrB), and the 60- and 69-kDa subunits of the vacuolar H+-ATPase, using polyclonal or monoclonal antibodies (Fig. 1E-H; C. Raymond, I. Howald, and T. Stevens, unpublished results).

Solutionsfor lmmunofluorescenceMicroscopy of Yeast Cells TEB: 200 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1% 2-mercaptoethanol; prepare immediately before use SPM: 1.2 M sorbitol, 50 mM potassium phosphate, pH 7.3, 1 mM MgCI2 Fixative (4% formaldehyde): To prepare 50 ml, add 2 g paraformaldehyde to 50 ml of water and heat. Add 350/A of 6 N NaOH and stir until the solution clears. Remove from heat and add 0.68 g of KH2PO 4 and 50/zl of I M MgC12 (the pH of the solution should be approximately 6.5). Always prepare immediately before use Polylysine: 1 mg/ml polylysine (Mr 400,000) in water;, stable for months at 4 ° 3o C. Wittenberg, S. L. Richanison, and S. I. Reed, J. Cell Biol. 105, 1527 (1987). 31 j. p. Ads and G. Blobel, J. CellBiol. 107, 17 (1988). 32 j. V. Kilmartin and A. E. M. Atlam.% J. CellBiol. 98, 922 (1984).

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Mounting medium: To prepare 5 ml, mix 4.5 ml of 100% glycerol, 500/d 10 mg/ml p-phenylenediamine (in 1 X PBS, pH 9), and 0.1 #1 DAPI (4',6'-diamidino-2-phenylindole, 1 mg/ml in water; DAPI stains nuclei with fluorescence in the blue range, and is routinely included). Mounting medium should be stored at - 20 ° or - 80 ° in the dark; discard the solution when it turns dark brown PBS-BSA: 5 mg/ml bovine serum albumin (BSA) in 1 × PBS plus 5 m M sodium azide as preservative Adsorption of Affinity-Purified Antibodies to Fixed Yeast Cells. Harvest approximately 1 × 10m° cells lacking the gene product of interest and prepare the cells for immunofluorescence as described below, except scale up the procedure 50- to 1oo-fold. Collect the cells by centrifngation and resuspend in 20 ml of PBS-BSA. Gently shake at room temperature for 30 rain, then divide the suspension into two tubes and collect the cells. Resuspend one-half of the cells in 500/d of PBS-BSA and add 2 0 - 5 0 / d of approximately 1 mg/ml affinity-purified antibody. Incubate at room temperature for 1 hr with gentle agitation. Centrifuge, remove the antibody solution, and use it to resuspend the other half of the fixed cells. Wash the first cell pellet with 500/zl PBS-BSA and save the solution. Repeat the 1 hr incubation with the second cell pellet. Remove the fixed cells from the adsorbed antibody preparation by centrifugation, wash the second cell pellet with the 500/~1 PBS-BSA wash from above, pool the supernatant solutions, and repeat the centrifugation once more to remove any remaining debris. Use immediately or store at 4°. Most adsorbed antibody preparations are stable at 4 ° for months. Although nonspecitic antibodies are removed by this procedure, substantial losses in specific antibody titer may occur. The optimal antibody dilution for immunofluorescence experiments must be determined empirically, but dilutions of 1 to 10 are typically used in our laboratory. Fixation of Cells for Immunofluorescence Microscopy. Grow 10 ml of yeast cells in YEPD or selective medium to a density of about 1 × l0 T cells/ml. Add 1.2 ml of 37% formaldehyde (commercially available) to the culture and incubate for 1 hr at 30°. Harvest the cells by centrifngation and resuspend in 2 ml of 1 × fixative. Shake gently at room temperature or 30 ° for about 16 hr (we routinely fix cells overnight at either temperature with good results). Harvest the cells, resuspend in 1 ml TEB, and shake gently at 30 ° for 10 rain. Centrifuge and resuspend the pellet in 1 ml SPM. Remove the cell walls by adding 25/zl of Glusulase (Du Pont NEN Products, Boston, MA) and 15/~1 of 10 mg/ml Zymolyase 100T (ICN Immunobiologicals, Lisle, IL; freshly prepared in SPM) and shaking gently at 30 ° for 30-60 rain. Pellet the spheroplasted fixed cells, wash once in 1 ml of 1.2 M sorbitol, and treat with SDS by resuspending in 5OO/zl of 1.2 M sorbitol and adding 5OO/d 1.2 M sorbitol plus 4% SDS. After a 2-rain

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incubation, harvest the cells by centrifugation at slow speed (e.g., 30 sec at 4000 g) and wash gently 2 times with 1-ml aliquots of 1.2 M sorbitol. Resuspend in 1-2 ml of 1.2 M sorbitol. The cells are now ready to be applied to a microscope slide. Notes. As discussed previously, the morphology of the vacuole is influenced by the way the cells are treated prior to fixation. The protocol just described involves fixing the cells directly in glucose-containing medium and thus many of the cells will contain several vacuoles. Also, cells with small buds will exhibit vacuolar segregation structures, in which the vacuole appears as an extended tubular structure that extends from the mother cell into the bud 2~' (Fig. IE,F). Modifying the procedure to include washing cells in glucose-free buffers prior to fixation will result in most of the cells containing a single, large vacuole (Fig. 1G,H). We fix cells for much longer periods of time than conventional protocols, z5 and this has greatly improved the morphological preservation of cells and the subcellular structures within them. Spberoplasted fixed cells are quite fragile and should be treated gently. Damaged cells have a squashed, fuzzy appearance when viewed under the microscope. We generally use fixed cells immediately after preparation and have little experience with their stability in storage. SDS treatment is required in order to achieve staining with certain antibodies (e.g., polyclonal rabbit anti-alkaline phosphatase), but the staining with other antibodies deteriorates with extended SDS treatment (e.g., rabbit polyclonal or mouse monoclonal anti-60-kDa vacuolar H+-ATPase subunit). The optimal concentration of SDS (1 -5%) and time of exposure (0.5 -5 rain) must be worked out empirically. Treatment of fixed cells with SDS makes the cells very adherent to polylysine-coated microscope slides (see Antibody Staining of Fixed Cells below). This adherence is a distinct advantage as most untreated cells can be lost during the extensive washes required for antibody treatment. Carefully controlled experiments have shown that SDS treatment does not change the distribution of vacuolar staining patterns observed, nor does it exert adverse effects on cell morphology. Antibody Staining of Fixed Cells. Prepare polylysine-coated slides by placing 10 gl of I mg/ml polylysine in each well of an 8-well multitest slide (Flow Laboratories, Inc., McLean, VA; Cat. No. 60-408-05). Make sure the solution covers the entire glass surface of the well. After I rain, aspirate the wells, wash each 5-6 times with 20 gl of water, aspirate again, and allow the slides to dry. Apply 40 gl of a freshly prepared fixed cell suspension to each well and allow to settle for 10 rain. Aspirate the fluid and wash each well 3 times with 20 #1 of PBS- BSA. Apply 20 gl of PBS- BSA and incubate for 30 rain at room temperature in a humid chamber (a petri dish

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containing a damp paper towel works well). Never allow the cells to dry out. Aspirate the wells and apply 10 ~tl of the primary antibody solution diluted appropriately in PBS-BSA. A I to 10 dilution of adsorbed primary antibody is typically used. Incubate the sfide in a humid chamber for 1 hr at room temperature. Aspirate the primary antibody solution and wash each well 6 times with PBS-BSA. Apply the secondary antibody solution and repeat the incubation as above (for amplification of signal, see below). After removal of the last antibody solution, wash the wells at least 9 times. Place small drops of mounting medium in the wells, cover with a 24 × 60 mm coverslip, and seal the ends of the coverslip to the slide with fingernail polish. Slides can be viewed immediately or stored at - 2 0 ° for weeks (Fig. 1E-H). Notes. In cases where the antigen being detected is abundant, fluorochrome-conjugated second antibody is applied directly. For proteins of low abundance, this two-step incubation procedure often yields a signal that is too weak to detect above the inevitable slight background fluorescence. To improve the signal-to-noise ratio, an antibody amplification procedure has been used.24b,2s which involves decorating the primary antibody with goat anti-rabbit IgG, followed by treatment with rabbit anti-goat IgG, followed finally by goat anti-rabbit IgG conjugated to the appropriate fluorochrome. In theory each bound antibody binds more than one antibody in the subsequent incubation, thus amplifying a weak signal. In practice, one observes a greater increase in vacuolar versus background fluorescence; however, the procedure is limited by the specificity of the primary antibody. All incubations with secondary antibodies should be done as described above. This procedure has given us better amplification of signals than procedures that use biotinylated second antibodies followed by fluorochrome-conjugated streptavidin that have been described elsewhere.25 We have had good results using secondary antibodies, reconstituted to l mg/ml, from both Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and Organon Teknika-Cappel Laboratories (West Chester, PA). Fluorochrome-conjugated secondary antibodies are used at l to 100 dilutions. When the amplification procedure is used, 1 to 1000 dilutions of intermediate secondary antibodies are used. Purification of Vacuoles

Several methods are available for purification of the yeast vacuole, all of which take advantage of the low buoyant density of the vacuole?TM Our method of choice, which is a modified version of the procedure used by

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Kakinuma et al., 35 involves enzymatic removal of the cell walls, osmotic lysis of the spheroplasts (without lysis of the vacuoles), followed by flotation of the vacuoles on a discontinuous Ficoll gradient. Other methods use polybase (e.g., DEAE-dextran) induced lysis of spheroplastsH,u or lysis of spheroplasts under isotonic conditions followed by density gradient centrifugation.33,34The procedure that follows has consistently given an 10-20% yield of vacuoles and low contamination of other organelles. Solutions

Zymolyase solution: 50 mM Tris-HC1, pH 7.7, 1 mM EDTA, 50% glycerol, 400 units (U)/ml Zymolyase 100T (4 mg/ml; ICN Immunobiologieals) Buffer A: 10 mM 2-(N-morpholino)ethanesulfonic acid (MES)/Tris, pH 6.9, 0.1 mMMgCI2, 12% Ficoll 4OO Buffer B: 10 mM MES/Tris, pH 6.9, 0.5 mM MgC12, 8% Ficoll 400 Buffer C (1X): 10 mM MES/Tris, pH 6.9, 5 mM MgCI2, 25 mMKC1 Cell Growth and Spheroplast Formation

Grow yeast cells to mid-log phase and collect 4 × 10~° cells (i.e., 4000 OD~o units) by centrifuging at 4400 g for 5 rain. Wash the cells once with distilled water at room temperature, and convert the cells to spheroplasts by resuspending in 100 ml of I Msorbitol and then adding 1 ml of Zymolyase solution. Shake the culture gently at 30* for 60-90 rain and cheek for completion of spheroplasting by diluting aliquots 10-fold into both buffer A and 1 M sorbitol; after about 5 rain at room temperature, the buffer A suspension should be much less opaque than the sorbitol suspension owing to lysis of the spheroplasts. Spheroplast L ysis

Collect the spheroplasts by centrifuging at 2200 g for 5 rain, and wash twice in 1 M sorbitol. Lyse the spheroplasts by resuspending the final pellet in 25 ml of buffer A and homogenizing at 0". Centrifuge the lysate at 2200 g for 10 rain at 4" to remove any unlysed spheroplasts, and save the supernatant. Save 2% of the spheroplast lysate for assessment of yield and purity. 33A. Wiemken, in "Methods in Cell Biology" (D. M. Pre"~ott, ed.), Vol. 12, p. 99. Academic Press, London, 1975. 34A. Wiemken, M. Schellenberg, and K. Urech, Arch. MicrobioL 123, 23 (1979). 35y. Kakinuma, Y. Ohsumi, and Y. Anraku, J. Biol. Chem. 256, 10859 (1981). M. Drift, T. Boiler, and A. Wiemken, Arch, Microbiol. 105, 319 (1975).

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Vacuole Flotation All subsequent manipulations are carried out at 0"-4"~ Transfer the superuatant to a polyallomer tube of appropriate size for a Beckman SW 28 rotor. Carefully overlay with about 13 ml of buffer A and centrifuge at 60,000 g for 30 min. Collect the white wafer floating on top of the Ficoll and homogenize in 6 ml of buffer A. Transfer the suspension to a polyallomer tube appropriate for a Beckman SW 41 Ti rotor. Overlay with 6 ml buffer B, then centrifuge at 60,000 g for 30 min. Collect the white wafer at the top of the tube and resuspend in a small volume (0.2-1 ml) of 2 X buffer C. Homogenize the purified vacuolar vesicles by passing the suspension up and down through a micropipette tip several times, then add an equal volume of 1X buffer C. Homogenization in buffer C causes fragmentation of the vacuoles into vacuolar vesicles, which may result in some loss of lumenal content. Store the vacuolar vesicles at - 80 ° Notes. The procedure just described usually yields 1- 3 nag of vacuolar protein, corresponding approximately to a 10-20% recovery of vacuolar vesicles as determined by vacuolar membrane marker assays (see below). The yield obtained may vary depending on the yeast strain used. It is important to proceed through the purification protocol as quickly as possible to avoid extensive loss of enzymatic activity and protein integrity (the vacuole, as noted above, contains a large proportion of the cellular hydrolases). Also, it is crucial to keep the sample cold during the last two steps of the purification, including precooling the swinging buckets and rotor for the ultracentrifuge runs. Collection of the floated vacuolar wafers can be very ditficult, especially for strains that give low yields of vacuoles. We generally use a spoonshaped stainless steel spatula that has been prewetted in buffer A to scoop up the vacuoles.

Determination of Yield: Vacuolar Enzyme Marker Assays There is an abundance of hydrolytic activities which are specific to the vacuole and are easily assayed. Several of these enzyme markers are soluble proteases such as carboxypeptidase Y, proteinase A (PrA), and proteinase B; the assays for these enzymes are described elsewhere in this volume.37 Our laboratory commonly measures three vacuolar membrane-associated activities for determination of yield: H+-ATPase, dipeptidyl aminopeptidase B, and ot-mannosidase. Protein determination is performed according 37 E. W. Jones, this volume [31].

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to Lowry et al. 3s except that 2% SDS is included in order to release all the lumenal proteins. Vacuolar H+-A TPase. The activity of the vacuolar H+-ATPase is monitored by a coupled enzyme-ATP regeneration assay system as described by L6tscher et al.39 with several modifications. Vacuolar vesicles (5I00/d) are assayed in I ml of a solution containing 25 m M Tris-acetate, pH 7.0, 25 m M KC1, 5 m M MgCI2, 2 m M phosphoenolpyruvate, 2 m M ATP, 0.5 m M NADH, 30 units of L-lactate dehydrogenase, and 30 units of pyruvate ldnase at 30 °. The assay is performed by adding the vacuolar vesicles directly to a cuvette containing the other components, then immediately observing the change in absorbance at 340 nm using the time drive mode on a spectrophotometer. Absorbance readings are linear up to an Am value of 3.0. The molar extinction coefficient for NADH (e) is 6.22 mM -n cm-l, and depletion of NADH is directly correlated to ATP hydrolysis. Specific activity corresponds to micromoles ATP hydrolyzed per minute per milligram protein. The vacuolar H+-ATPase is insensitive to 2 mM sodium azide and 0.1 mM sodium vanadate, distinguishing it from the mitochondrial and plasma membrane ATPases, respectively. The yeast vacuolar H+-ATPase,2° like other vacuolar H+-ATPases,4° is specifically inhibited by the macrolide antibiotic bafilomycin A~ at very low concentrations (/50 = 0.6/lg bafilomycin A J m g vacuolar vesicles);2° unfortunately, this drug is not commercially available at this time. Dipeptidyl Aminopeptidase B. The following assay was adapted from Suarez Rendueles et al. 4~ To a 200-/,1 sample containing 20-100/zl of vacuolar vesicles and 0.5% Brij 58, add 250/~1 ofHEPES/Tris, pH 7.0, and 50/~1 of 3 m M alanylprolyl-p-nitroanilide (Bachem Biochemicals, Switzerland; prepare in 25% methanol and store at 4°). Incubate at 37 ° for 1 hr, and stop the reaction by adding 500 ~1 of 5% ZnSO4 and 100/~1 of 7.5% B a ( O H ) 2 " 8 H 2 0 ( m u s t boil to dissolve; store in a flask with a soda lime tube trap at 25 °). Pellet the particulate matter in a clinical centrifuge at full speed for 5 min, and then read the absorbance of the optically clear supernatant at 405 nm. Readings are linear up to an A4os value of 0.7. For whole cell lysate determinations, cells must first be collected and then washed in 0.9% NaC1. Freeze the washed pellet at - 8 0 ° for at least 10 min. After thawing on ice, resuspend the cells in 50 mM potassium acetate, pH 5.0 (use 5/~1 per OD6oo unit of cells). Add 5% Brij 58 to a final ss O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 39 H. L6tscher, C. deJong, and R. A. Capaldi, Biochemistry 23, 4128 (1984). (o E. J. Bowman, A. Siebers, and K. Altendorf, Proc. Natl. Acad. Sci. U.S.A. 85, 7972 (1988). 41 M. P. Suarez Rendueles, J. Schwencke, N. Garcia Alvarez, and S. Gascon, FEBS Lett. 131, 296 (1981).

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concentration of 1% and leave on ice for 30 rain. Increase the sample volume to 200/tl in 0.5% Brij 58 and assay as described above. Yeast cells contain two dipeptidyl aminopeptid_a_c~s, DPAP A and DPAP B.42 DPAP A is heat stable and is localized to the secretory pathway, where it processes the mating pheromone, ¢x-factor. DPAP B is situated in the vacuolar membrane6,43 and is sensitive to heat treatment. To distinguish between the two DPAP activities, divide the sample to be assayed in half and incubate one tube at 60* for 15 rain, followed by 5 rain on ice. DPAP B activity corresponds to the difference between the A4o5 readings of the heat-treated and untreated samples. ol-Mannosidase. ¢x-Mannosidase activity is assessed as described.~ Mix 0.6 ml of 0.1 M MES/NaOH, pH 6.5, plus 0.2% Triton X-100 and 0.2 ml of 4 m M p-nitrophenyl-~-D-mannopyranoside with 25 - 200/zl of vacuolar vesicles and adjust the final reaction volume to 1 ml with water. Incubate at 37* for 1 hr and stop the reaction by adding 1 ml of 0.5 M glycine/ Na2CO 3, pH 10.0. Measure the absorbance at 400 nm. Readings are linear up to an A4o0 of 1.5. The molar extinction coefficient for p-nitrophenol is 16 m M - l em -l. Assessment of Purity." Nonvacuolar Marker Enzyme Assays

The extent of contamination of the isolated vacuoles by nonvacuolar proteins can be determined by assaying marker enzymes of other subcellular compartments. We routinely check glucose-6-phosphate dehydrogenase (cytoplasm), NADPH-cytoehrome-c reductase (endoplasmic retieulum), azide-sensitive ATPase (mitochondria), and vanadate-sensitive ATPase (plasma membrane). We have not checked for nuclear contamination, although we expect that it is very low. More information on marker assays for different subcellular compartments is provided elsewhere in this volume. 45 Cytoplasm: Glucose-6-Phosphate Dehydrogenase. Glucose-6-phosphate dehydrogenase is a commonly measured cytoplasmic activity.~ Resuspend the sample in 1.6 ml of 50 m M Tris-HCl, pH 7.6, and 0.2 ml of 5 m M NADP + in 1% NaHCO3. Incubate at room temperature for 5 rain, then read the A~o of one-half the mix if--0). Immediately add 0.1 ml of 6.67 m M glucose 6-phosphate to the other half of the mix to initiate the 42D. Julius, L. Blair, A, Brake, G. F. Sprague,Jr., and J. Thorner, Cell (Cambridge, Mass.) 32, 839 (1983). 43C. Bordallo,J. Sehweneke,and M. S. SuarezRendueles,FEBS Lett. 173, 199 (1984). D. J. Opheim,Biochim. Biophys. Acta 524, 121 (1978). 4sA. Franzusoff,J. Rothblatt, and R. Sehekma~,this volume[45]. G. W. L6hr and H. D. Wailer, in "Methodsof EnzymaticAnalysis"(H. U. Bergmeyer, ed.), 2nd Ed., Vol. 2, p. 636. AcademicPress, New York, 1974.

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reaction. Incubate at room temperature for 15 rain then read the A34o. Readings are linear up to an A m of 0.5. The molar extinction coefficient for NADH is 6.22 m M -~ cm -~. The level of contamination by glucose 6phosphate in purified vacuoles is typically very low (i.e.,

Methods for studying the yeast vacuole.

644 CELLBIOLOGY [44] [ 4 4 ] M e t h o d s for S t u d y i n g t h e Y e a s t V a c u o l e By CHRISTOPHER J. ROBERTS, CHRISTOPHER K. RAYMOND, C...
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