Isolation and Identification of the Cytoplasmic Membrane

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p Vol. 36, No /78/ $02.00/0 Copyright C 1978 American Society for Microbiology Printed in U.S.A. Isolation and Identification of the Cytoplasmic Membrane from Saccharomyces carlsbergensis by Radioactive Labeling MICHAEL J. LEWIS* AND PURUSHOTTAM C. PATEL Department of Food Science and Technology, University of California, Davis, California Received for publication 28 September 1978 The cytoplasmic membrane of Saccharomyces carlsbergensis was isolated by enymatic digestion of the yeast cell wall, followed by lysis of the protoplasts and fractionation by ultracentrifugation in a discontinuous sucrose density gradient. Location of the cytoplasmic membrane fraction on the sucrose gradient was made by labeling intact protoplasts with [G-3H]dansyl chloride, and was settled at the 50% (wt/vol) sucrose gradient (d = g/cm3). Approximately 80% of the radioactivity was found in the membrane fraction prepared in the presence of Mg2" ions. However, when protease inhibitors were used in the preparation step, the membrane fraction contained over 90% of the total radioactivity. The presence of Mg2e ions during membrane isolation and purification enhanced the aggregation of membrane components but, at higher concentrations, as well as in the prolonged presence of Mg2e ions in the membrane suspension, it caused the breakdown of membrane components. The membrane preparation contained Mg2+-adenosine triphosphatase, which was insensitive to oligomycin and ouabain. The distribution of Mg2 -adenosine triphosphatase in different fractions during sucrose gradient is reported. 851 Biochemical knowledge of membrane structure is primarily based on studies of easily isolated membranes, such as membranes of human erythrocytes (4, 11), the naturally occurring microbial L-forms (Mycoplasma) (5), and Halobacterium (3). The structure and function of cytoplasmic membranes are the subject of many investigations in biology, all of which depend on the development of methods for the isolation of pure membrane preparations. Few studies have been carried out on the cytoplasmic membrane of yeast. Longley et al. (9), working with Saccharomyces cerevisiae, and Garcia-Mendoa and Villanueva (6) and Schwencke et al. (J. Schwencke, J. Krsulovic, M. Rojas, and G. Farias, Abstr. Third Int. Symp. Yeast Protoplasts, p. 90, 1972), working with Candida utilis, made membrane preparations from protoplasts, after enymatic digestion of the cell wall, followed by osmotic lysis of the protoplast and, finally, the isolation of membranes by differential centrifugation of the lysates. Nurminen et al. (14) also studied the plasma membrane fragments during onal centrifugation of homogenates from glucose-repressed S. cerevisiae. The numerous methods used to isolate the cytoplasmic membrane and the criteria used for assessing purity render it impossible to compare data from different laboratories using different yeasts. One of the most successful methods employed to locate and assess the purity of the cytoplasmic membrane preparation has involved radioactive labeling of the surface of intact protoplast and observing the distribution of radioactivity upon subsequent fractionation (17, 12). That technique has been applied to the yeast form and the mycelial form of C. albicans. This paper presents a method for isolating a highly pure preparation of cytoplasmic membrane of a brewing yeast; labeling with radioactive dansyl chloride is used to locate this membrane. In addition, the effect of inhibitors and Mg2" ions on membrane aggregation and breakdown of membrane components has been investigated. MATERIALS AND METHODS Organism and growth. S. carlsbergensis (University of California no ), a flocculent commercial brewer's yeast, was grown as previously reported (8), harvested in exponential phase, and converted into protoplasts by using the glusulase (Endo Laboratories) and dithiothreitol treatment as previously described by Sommer and Lewis (18). Radioactive labeling. Yeast protoplasts were suspended in 10 ml of 0.8 M mannitol phosphate buffer (ph 9.2) before addition of radioactive dansyl chloride. A 20-,u amount of dansyl chloride having a specific radioactivity of 17 Ci/mmol in benene ([G-3H]dansyl chloride, 1.0 mci/ml, is prepared from 1-dimethylaminonaphthalene-5-sulfonic acid; Amersham/Searle Corp.) was added slowly into protoplast suspension

2 852 LEWIS AND PATEL with gentle shaking and allowed to react for 20 min at room temperature. Radioactive-labeled protoplasts were then harvested by low-speed centrifugation (755 x g) and washed three times in the mannitol phosphate buffer. Osmotic lysis of protoplasts. Protoplasts were lysed in a large volume of 50 mm phosphate buffer (ph 6.7), with or without added 10 mm MgCl2. The ghost membranes were collected by centrifugation at 3,000 x g for 10 min at 4 C and washed three times in 10 mm tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (ph 8.0). Discontinuous sucrose density gradient. For further purification of the membrane, a discontinuous sucrose gradient was prepared by layering, in a 60-ml cellulose nitrate centrifuge tube, the following sucrose solutions, made up in 10 mm Tris-hydrochloride buffer (ph 8.0) with or without added 10 mm MgCl2: 60% (d = 1.224), 50% (d = 1.186), and 25% (wt/vol) sucrose (d = 1.093); each solution was 16 ml (Fig. 2). A dense suspension of ghost membranes was layered on the top of the gradient and then centrifuged in a Beckman model L ultracentrifuge at 75,000 x g (Ray) for 3 h at 4 C, using an SW 25.2 swinging bucket rotor. Ten crude fractions were collected from the top by a syringe or dropwise fractions collected by puncturing the bottom of the centrifuge tube. The membrane fraction, which sedimented at the interface of the 50 and 25% sucrose solutions, was collected and washed three times in 10 mm Tris-hydrochloride buffer (ph 8.0). Radioactivity measurement. The total radioactivity of the crude fractions was determined by using a Beckman LS 3145P model scintillation counter in 10 ml of scintillation liquid (complete cocktail liquid, 3a70B, Research Products International). Mg2 -ATPase and protein estimation. Mg2l-dependent adenosine triphosphatase (ATPase) was assayed as described previously (16). Protein was determined by the method of Lowry et al. (10), using bovine serum albumin as standard. RESULTS AND DISCUSSION Cytoplasmic membrane fractionation. Figure 1 depicts the flow chart of isolation of the cytoplasmic membrane on a discontinuous sucrose gradient. Upon ultracentrifugation of ghost membrane on sucrose gradient, the pattern of fractions was roughly constant, and they were designated as F1 through Flo (Fig. 2). The fraction (F1) on the top of the gradient consisted of lipid material, which might have been loosely associated with the membranes and freed during the gradient centrifugation. However, if 10 mm MgCl2 ions were included in the sucrose gradient or in the sample suspension, then this band disappeared. The bulk of the membrane formed a tight band (F5) at 50% sucrose gradient level, having a density of g/cm3 (17). Through intermixing of sucrose solutions at both interfaces of the membrane containing solution, a density distribution was created which separated the membranes by two clear ones from the light bands appearing above (F4) and below APPL. ENVIRON. MICROBIOL. (F6) the membrane. The bottom pellet of the sucrose gradient contained intact cells, whole spheroplasts, and cell debris (Flo). Identification of cytoplasmic membrane. To identify the membrane fraction on the sucrose gradient, the surface of the intact protoplast was labeled with radioactive dansyl chloride. This is a nucleophilic reagent, and dansylation of protein is favored by alkaline condition. The specificity of the reagent in labeling the cytoplasmic membrane and not the intracellular membranes probably depends on the differences in ph values between the suspending buffer and the cell cytoplasm, since the cytoplasmic membrane is exposed to dansyl chloride (2). The results of the experiments in which the protoplast membrane was dansylated before lysis and fractionation on sucrose gradient are shown in Fig. 3. The material from fraction F5 contained the highest total radioactivity (85%), with small amounts of the radioactivity distributed in other fractions. Similarly, if only 0.1 ml of ghost membrane suspension was layered on an identical gradient to that in Fig. 3 and small fractions were collected dropwise through the bottom of the centrifuge tube, then a similar pattern of radioactive distribution was found (Fig. 4). The highest labeling of radioactivity was found at the same position in both experiments. This confirms that our method of collecting the fractions with a syringe (Fig. 3) is appropriate. Moreover, acid phosphatase, which is associated with the yeast wall, was not detected in the purified preparation, nor was the mitochondrial enyme, cytochrome c oxidase. The absence of these enymes shows a lack of contamination with cell wall fragments and mitochondrial membranes in this preparation. Specimens of the membrane viewed with an electron microscope (L.C.A. EMU-3G electron microscope) had the typical trilaminar structure at the site of the protoplast rupture. M&P+-ATPase. To further identify the mem- we assayed Mg2e-dependent brane band (F5), ATPase in all fractions (Fig. 5). When calculated in terms of total ATPase activity, fraction 5 contained about 90% of the activity. However, specific activity was 11.9 ttmol of inorganic orthophosphate per mg of protein per h in this fraction. The presence of mitochondrial Mg2+- ATPase is unlikely, because the yeast was grown in a glucose-repressed medium. The mitochondrial Mg2e-ATPase, however, differs from Mg2+- ATPase of the plasma membrane in being oligomycin sensitive (15). In this context, we tested Mg2+-ATPase activity in the cytoplasmic membrane fraction in the presence of oligomycin and ouabain and found it absolutely insensitive.

3 YEAST CELLS E!NZYMATIC DIGESTION OF CELL WALL by glusulase and dithiothreitol treatment at 370C Centrifugation at 4,000 x g, 10 min, 4 C Supematant discarded PROTOPLAST FORMATION' Stabilied in 0.8 M mannitol phosphate buffer RADIOACTIVE LABELING by [G-3H]dansyl chloride Centrifugation at 755 x g, 10 min, 4 C Supernatant discarded 4 [ LABELED PROTOPLASTS in 0.8 M mannitol phosphate buffer Supernatant discarded l OSMOTIC LYSIS OF PROTOPLASTS Centrifugation at 3,000 x g, 10 min, 40C LYSED GHOST MEMBRANE DISCONTINUOUS SUCROSE DENSITY GRADIENT Centrifugation at 75,000 x g, 3 h, 40C with Beckman L SW 25.2 Rotor CYTOPLASMIC MEMBRANEJ FIG. 1. Flow chart for isolation of cytoplasmic membrane from yeast S. carlsbergensis. FRACTION NUMBER SAMPLE-9, F, 25% F2 sucrose CENTRIFUGATION 50% 9 6 stcros 75,000xg,3h,4C F7 60% F9 sucrose - n - i/ 14&ov FIG. 2. Schematic diagram offractionation ofghost membrane on discontinuous sucrose density gradients. 853

4 854 LEWIS AND PATEL APPL. ENVIRON. MICROBIOL. o 100 C..) 80 _ Rodiooctivity c=~proteiin O20 Og F41 F3 J F2 FI 60%. 50% 25% sucrose sucrose sucrose [BOTTOM] DISCONTINUOUS SUCROSE GRADIENT (TOP] FIG. 3. Percent distribution of radioactivity and protein upon fractionation of the ghost membrane prepared from radioactively labeled protoplast from S. carlsbergensis. Fractions were collected from the top of the gradient. Crosshatched area shows where cytoplasmic membrane was settled. Aggregation and breakdown of membrane components. Marriott (13) prepared plasma membranes after labeling protoplasts of C. albicans and presented evidence that fractionation on a single discontinuous gradient yields a product substantially contaminated with other cell components. He suggested that this is probably due to the entrapment of membrane vesicles. Second and third centrifugations are required to give a pure preparation. In our study, we observed that Mg2" caused aggregation of membrane proteins and thus obtained higher radioactivity in the membrane fraction than without Mg2e ions in the preparations. However, prolonged contact of Mg2" ions with the membrane suspension resulted in a greater breakdown of membrane components rather than aggregation. Similar findings were reported by King and Morrison (7) with Ca2" ions on human erythrocyte membrane proteins. To control the protease activity which may have caused the hydrolysis of membrane proteins, iodoacetate and phenylmethyl-sulfonyl fluoride (final concentrations, 0.01 M and 1.5 mg per ml, respectively) were added as inhibitors to the ghost membrane suspension. Discontinuous sucrose density gradient followed (Fig. 6a). Frac- H 60% - 50% 25% sucrose sucrose sucrose SAMPLE [ BOTTOM] DISCONTINUOUS SUCROSE GRADIENT (TOP] FIG. 4. Fractionation of the ghost membranes prepared from radioactively labeled protoplast from S. carlsbergensis. Fractions were collected dropwise from the bottom of the gradients and measured for total radioactivity (BOTTOM] K 60% 50%- 25%-4fsucrose sucrose sucrose DISCONTINUOUS SUCROSE GRADIENT (TOP] FIG. 5. Distribution ofmg2+-atpase activity upon the fractionation of the ghost membranes on discontinuous sucrose gradients. Fractions were collected from the top of the gradients. Crosshatched area shows where cytoplasmic membrane was isolated.

5 VOL. 36, 1978 < 00_ La.. 80 _ Rodiooctivity oi _ oprotein QC.60 - J a 71 F861f7 1F6_ F+ F21, -60%_-O 50%25%% sucrose sucrose sucrose < (BOTTOM] DISCONTINUOUS SUCROSE GRADIENT (TOP] 0 cr I~- S1.- i_ m o I b _ Rodiooctivity Protein -60% 6 50% - 25% sucrose sucrose sucrose [BOTTOM] DISCONTINUOUS SUCROSE GRADIENT [TOP] FIG. 6. Effect of inhibitors on the isolation of the cytoplasmic membrane ofyeast during discontinuous sucrose gradient. Fractions were collected from the top of the gradients after (a) first centrifugation and (b) second centrifugation. CYTOPLASMIC MEMBRANE OF YEAST 855 tion F5 (cytoplasmic membrane) contained a very high level of labeled radioactivity (93%), indicating a very low degree of breakdown of membrane components in the presence of inhibitors. Although the pattern of protein distribution was not similar to the radioactivity distribution, when a second discontinuous sucrose gradient was performed the redistribution of both the protein and the radioactivity (93%) was found to be very similar (Fig. 6b). It appears, therefore, that there was no further purification and, furthermore, that there was very little breakdown of membrane components. This observation suggests that a single sucrose gradient is sufficient to achieve a rather pure (93%) membrane preparation. A second or third centrifugation will lead to the breakdown of membrane components unless some precautions are taken. The use of specific reagents for labeling cytoplasmic membranes from other sources is now well established (1). Schibeci et al. (17) used various radioactive compounds to label yeast protoplasts and monitored the distribution of radioactivity upon sucrose gradient fractionation. Thus, the results provided in this paper indicate that preparations of cytoplasmic membranes from the brewing yeast S. carisbergensis were devoid of much contamination from other cell components. In addition to the pattern of distribution of radioactive label and protein after centrifugation, further evidence for purity includes the high level of Mg2+-ATPase, which was absolutely insensitive to oligomycin and ouabain. 1ITERATURE CITED 1. Bretscher, M. S Membrane structure: some general principles. Science 181: Bretscher, M. S On labeling membranes. Nature (London) New Biol. 245: Brown, A. D The cation sensitive dissolution of the cell membrane of the halophilic bacterium, Halobacterium halobium. Biochim. Biophys. Acta 75: Dodge, J. T., C. Mitchell, and D. J. Hanahan The preparation and chemical characteristics of hemoglobin-free ghost of human erythrocytes. Arch. Biochem. Biophys. 100: Engelman, D. M., T. M. Terry, and H. J. Morowit Characteriation of the plasma membrane of Mycoplasma laidlawii. I. Sodium dodecyl sulfate solubiliation. Biochim. Biophys. Acta 135: Garcia-Mendoa, C., and J. R. Villaneuva Preparation and composition of the protoplast membrane of Candida utilis. Biochim. Biophys. Acta 135: King, L. E., Jr., and M. Morrison Calcium effects on human erythrocyte membrane proteins. Biochim. Biophys. Acta 471: Lewis, M. J., and H. J. Phaff Release of nitrogenous substances by brewer's yeast. III. Shock excretion of amino acids. J. Bacteriol. 87: Longley, R. P., A. H. Rose, and B. A. Knights Composition of the protoplast membrane from Saccharomyces cerevisiae. Biochem. J. 108: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin

6 856 LEWIS AND PATEL phenol reagent. J. Biol. Chem. 193: Maddy, A. H., and B. R. Malcom Protein conformations in the plasma membrane. Science 150: Marriott, M. S Isolation and chemical characteriation of plasma membranes from the yeast and mycelial forms of Candida albicans. J. Gen. Microbiol. 86: Marriott, M. S Mannan-protein location and biosynthesis in plasma membranes from the yeast forn of Candida albicans. J. Gen. Microbiol. 103: Nurminen, T., L. Taskinen, and H. Suomalainen Distribution of membranes, especially of plasma membrane fragments, during onal centrifugation of homogenates from glucose-repressed Saccharomyces APPL. ENVIRON. MICROBIOL. cerevisiae. Biochem. J. 154: Nurminen, T., L. Taskinen, and H. Suomalainen Distribution of plasma-membrane fragruents during onal centrifugation of homogenates from aerobic Saccharomyces cerevisiae. J. Gen. Microbiol. 98: Ohwaki, K., and M. J. Lewis Adenosine triphosphatase of Saccharomyces carlsbergensis. J. Inst. Brewing 77: Schibeci, A., J. B. M. Rattray, and D. K. Kidby Isolation and identification of yeast plasma membrane. Biochim. Biophys. Acta 311: Sommer, A., and M. J. Lewis Effect of dithiothreitol on yeast spheroplast formation and invertase release. J. Gen. Microbiol. 68: Downloaded from on January 18, 2019 by guest