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and 5 mg/ml BSA, by repeated low speedcentrifugation (50g). The inclusion of BSA was necessary because released cellular debris, most notably chloroplasts, ...
Plant Physiol. (1980) 65, 1053-1057 0032-0889/80/65/1053/05/$00.50/0

Labeling and Isolation of Plasma Membranes from Corn Leaf Protoplasts' Received for publication September 13, 1979 and in revised form January 2, 1980

DAVID S. PERLIN AND ROGER M. SPANSWICK Section of Botany, Genetics and Development, Division of Biological Sciences, Plant Science Building, Cornell University, Ithaca, New York 14853 MATERIALS AND METHODS Plant Material and Protoplast Isolation. Zea mays L., cv. A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confiwmed with the aid of an external Golden Bantam, was grown in a fertilized peat-Vermiculite soil label, diazotized 1I25lliodosulfanilic acid. Gentle cell disruption enabled mixture at room temperature under continuous cool-white fluinternal organelles to be maintained intact and thus facilitated separation orescent lighting. Protoplasts were isolated from the youngest fully from the plasma membrane. The plasma membrane-enriched fraction was expanded leaves of 12- to 14-day old plants by the method devoid of chloroplast or mitochondrial markers, whereas markers for the described by Earle et al. (3). All conditions were identical except endoplasmic reticulum and golgi indicated minimal contamination. The that the digestion enzyme was passed through a Sephadex G-50 highly enriched plasma membrane fraction contained a Mg2e-dependent, column prior to use and digestion was carried out in the dark KV-stimulated ATPase with a pH optimum near neutrality. The position of without shaking. Protoplasts were liberated by gently swirling the the membranes on sucrose density gradients indicates that the plasma digested tissue. Following isolation, the protoplasts were washed membranes have characteristics similar to other plasma membrane frac- five times in 0.6 M sorbitol, 50 mm K-phosphate buffer (pH 7.5), and 5 mg/ml BSA, by repeated low speed centrifugation (50g). tions. The inclusion of BSA was necessary because released cellular debris, most notably chloroplasts, would adhere to the protoplast surface. BSA eliminated this problem and had the added advantage of promoting greater total yield of intact protoplasts. The washed protoplasts were further purified by aqueous two-phase polymer separation (1 1) and resuspended in 0.6 M sorbitol and 50 mm phosphate buffer (pH 7.5) and stored on ice. Plasma membrane-enriched fractions have been isolated from acid of high specific radioactivity Labeling. numerous plant sources, including oat and corn roots (9, 10, 15), (greater than['25Illodosulfanilic was converted to its diazonium salt Ci/mmol) 1,000 corn coleoptiles (7, 12), green onion meristem (24), leek epidermal by the sequential addition of equal quantities of NaNO2 and HC1, cells (2), sugarcane leaves (21), sugarcane cell suspensions (23), as described in the instructions accompanying the labeling kit soybean suspension cultures (5), soybean roots (6), and tobacco supplied New England Nuclear. Unlabeled diazotized sulepidermis (16). In most cases, the isolation method employed fanilic acidbywas prepared in the same manner. The final concenrequired mechanical disruption, usually grinding, followed by tration of diazotized sulfanilic acid was 50 ,lM, and 60-80 ,uCi of differential and density gradient centrifugation. Although the were used per experiment. application of these procedures to plant plasma membrane isola- I[25liodine Washed protoplasts were incubated in the presence of label for tions has been widespread, the identity and purity of isolated by removal of the labeling material has not always been confirmed. This is due primarily to 30 min at 4 C. Labeling was terminated M 50 mm phosphate 0.6 sorbitol, with medium and replacement the lack of general acceptance for proposed intrinsic plasma membrane markers. Attempts to label the plasma membrane buffer (pH 7.5), and 10 mg/ml BSA. The additional protein was externally prior to isolation have met with limited success (5, 7, needed to complex loosely adsorbed, unreacted label. The protoplasts were washed at least five times in the above medium. 19, 22). Plasma Membrane Isolation. Labeled protoplasts were resusHarsh disruption procedures, although satisfactory in liberating plasma membranes from root or storage tissue, are undesirable pended in 0.6 M sorbitol and 50 mm phosphate buffer (pH 7.9). when applied to green tissue due to disruption of intact chloro- An equal volume of 50 mm Tris. Mes (pH 7.9), 2 mm Na2EDTA,2 plasts. The chloroplast fragments produced are difficult to separate and 6 mm DTT was added to the suspension. The protoplasts were from other membranes on the basis of differences in density or allowed to swell for 5 min causing them to become extremely sedimentation characteristics. To circumvent this problem, we fragile. Lysis was readily achieved by six gentle passes of a Teflon have used corn mesophyll protoplasts as a starting material for plunger in a glass Teflon hand-held homogenizer. This process resulted in a nearly complete lysis of intact protoplasts. The lysate plasma membrane isolations. Protoplasts exhibit two basic advantages over intact tissues. was layered on a 35% sucrose pad and centrifuged at 10,00Og for They can be lysed gently, preserving the intemal organelles intact 10 min (Fig. 1). Dense organelles (e.g. chloroplasts and mitochonand they can be labeled readily by extemal probes. Using both of dria) readily moved into the sucrose pad along with unbroken these characteristics, this study presents evidence for the isolation protoplasts and membrane aggregates. The supernatant fraction was then layered on a discontinuous sucrose gradient (31 and 38% of plasma membranes from green leaf tissue. ABSTRACT

2 'This research was supported by National Science Foundation Grants Abbreviations: Na2EDTA: ethylenedinitrilotetraacetic acid, disodium PCM 75-15277 and PCM 78-12119. salt. 1053

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PERLIN AND SPANSWICK

Table I. Distribution of the Diazotized ['25IJIodosulfanilic Acid Label in DifJerent Subcellular Fractions Total RaTotal . . Label Fraction a

Fraction E

Fraction |

AL

ysate

dioactivity resuspend

80,0009 30 min

35%

10,009t 80,00Og 10min 45min

59

scro"

38%j

C:

35,500 17,520 17,980 12,960 5,020

B C D E See Figure 1.

Recovered cpm/mg protein 1.1 x 103 1.48 x 103 1.05 x 103 23.1 x 103 0.32 x 103

17.1 0.56 16.3

ductase

FIG. 1. Diagram illustrating how each major subcellular fraction was obtained. Fractions were designated as follows (see under "Materials and Methods"): (A) lysate; (B) 10,000g pellet; (C) lO,OOOg supernatant; (D) final 80,000g pellet; and (E) fraction above the 31 and 38% sucrose interface in the first 80,000g centrifugation.

S

49 37 14

B

D

_-

ATased ATPase

ductase

junol/mg protein * h A

C

100

so

A

Protein mg 32.1 11.8

Table II. Activity of Major Organelle Enzyme Markers in the Various Subcellular Fractions Isolated The value in parentheses represents the total activity of the fracion. NADH NADPH tet K+-StimuFrcin c Rec ReCyt Cyt Oxidase IDPase

i

l

Fraction B

cpm

PELLET Fraction D

Fractionj

Plant Physiol. Vol. 65, 1980

a

0.68

(21.6)

1.37 (43.8)

1.26 (40.3)

1.32 (42.2)

0.38 (12.2)

1.5 (20.6)

0.89 (12.2)

0.66 (9.10)

0.82 (11.2)

0.75 (10.3)

1.71

1.69

(31.9)

(31.6)

1.70 (31.8)

(11.8)

0.04 (0.66) 0.20 (0.22)

0.31 (0.34)

0.27 (0.30)

0.00

1.76

(0.00)

(31.6)

1.76 (31.4)

1.41 (1.57)

0.63 6.60

(7.32)

a

E

60 _-

4

1.73

(031.1)

0.44 (7.92)

See Figure 1.

40 _

'The KV-stimulated component was determined by subtracting the activity obtained in the absence of K+ from the activity obtained in the presence of KV.

20 _

v

a

005

I

I

I

0 10

050

10

Diacotied Sulfanilic acid (mM)

FIG. 2. The effect of diazotized sulfanilic acid ATPase activity.

on

membrane-bound

sucrose) and centrifuged at 80,000g for 45 min. The interface (31/ 38) was removed with a pasteur pipette, diluted in 0.25 M sucrose and 10 mM Tris.Mes (pH 7.0), and pelleted at 80,000g. The pellet was resuspended and assayed. Enzyme Assays. Protein was determined by a modified Lowry procedure (18). ATPase activity was determined by the method described by Ames (1). All reductase assays were performed according to Hodges and Leonard (10), and latent IDPase activity was determined by the method of Ray et al. (20). RESULTS A primary concern during the labeling process was to prevent modification of specific membrane properties. Diazotized [12IJiodosulfanilic acid used at 50 ,uM did not significantly alter membrane-bound enzyme activity, particularly the K+-stimulated ATPase (Fig. 2). Once labeled, the plasma membrane could be followed easily through the isolation procedure. Table I indicates the presence of label through the fractionation process and demonstrates that the fraction with highest specific radioactivity came

Table III. Distribution of Labeled Chloroplasts and Total Chlorophyll in D/fferent Subcellular Fractions Chloroplasts were isolated from lysed protoplasts and collected in 0.3 M sorbitol and 50 mm phosphate buffer (pH 7.5). Labeling was identical to the methods used for protoplast labeling. Fraction' Chlorophyll

A B C

Total

Radioac-

tivity

mg/ml

cpm

0.35 2.51 NDb ND ND

88,500 84,400 5,750

D E a See Figure 1. b Not detectable.

120 5,000

Specific

Radio-

Label

Re-

activity

covered cpm/mg protein % of total

2,160 4,200 295 600 250

95.4 6.5 0.14 5.7

from recovery of the final 80,000g pellet (fraction D). The specific radioactivity of this fraction was much greater than that of any other fraction recovered. Analysis of this fraction for major organelie contamination indicated that it was essentially devoid of markers for the ER, mitochondria (Table II), and chloroplasts (Table III). Golgi contamination was also demonstrated to be minimal, yet it contributed proportionately more to the fraction than the other organelles. Fraction D contained the highest specific activity of K+-stimu-

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PLASMA MEMBRANE LABELING AND ISOLATION

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lated ATPase activity (Table II). This enzyme activity has been associated with plasma membranes from root and coleoptile tissue (6, 8, 9, 13-15). Further characterization of this fraction revealed that the enzyme activity had a clear pH optimum at pH 6.5 (Fig. 3), was stimulated by Mg2+, and was further stimulated by K+ (Table IV). Sucrose density centrifugation of fraction D showed that peak radioactivity corresponded with the peak of K+-stimulated activity but did not coincide with markers for major organelles (Fig. 4). Since mitochondria and plasma membranes reach sedimentation equilibrium at approximately the same density it was important to show that these two membranes were separate. This was done by contaminating the gradient with isolated mitochondria and comparing the peak of radioactivity to the peak of Cyt c oxidase activity. Figure 4 clearly demonstrates this separation. Thus, labeling and marker enzyme studies would indicate that the final 80,000g pellet (fraction D) was enriched with plasma membranes. In a further attempt to demonstrate the purity of the proposed plasma membrane fraction, we hypothesized that a small mitochondrial contamination might contribute significantly to Mg2+ATPase activity. In an effort to test this hypothesis, the ATPase 200

Fractions

15-0-

co

FIG. 4. Density gradient profile of the final 80,000g pellet (fraction D). Fractions enriched in the following markers: (A) NADH Cyt c reductase (0), latent IDPase (0); (B) radioactivity (0), Cyt c oxidase (0); (C) K+stimulated ATPase activity (0), per cent sucrose (0). Panels B and C were obtained by adding a portion of fraction B to fraction D and running the sample on a 15-45% linear sucrose gradient at 80,000g for 3 h. Panel A was obtained by pooling two l00,OOOg fraction E pellets and running the membranes on a separate linear sucrose gradient. The percent sucrose shown in panel C applies to panels B and C, but also closely approximates the positions shown in panel A.

E 0

E

.a10-0

50

o-oL 45/

5-5

65

75

85

pH

FIG. 3. A profile of Mg-ATPase activity as a function of pH in the presence and absence of added 50 mM KCI. KCI included (x), KCI excluded (0), and the difference between the two activities (0). Table IV. The Effect of Mg2 and K+ on Membrane-bound A TPase Activity Treatment Specific Activity ,.smol Pi/mg protein * h 3 mM ATP 6.49 3 mM ATP + 3mM MgSO4 11.2 3 mM ATP + 3mM MgSO4 + 50 mm KCI 16.4 3mMATP+50mm KCI 6.84

activity was measured as a function of pH in the presence and absence of oligomycin (Fig. 5). Oligomycin has been shown to be a potent inhibitor of the mitochondrial coupling factor ATPase. The plasma membrane ATPase activity was unaffected by this inhibitor and the validity of this test was clearly demonstrated by contaminating one fraction with mitochondria and reversing the effect with oligomycin. Thus, mitochondrial contamination appears insignificant and certainly does not contribute to the ATPase activity observed. One of the prime difficulties in using green tissue as a starting material for plasma membrane isolations is the problem of disrupting chloroplasts and producing small fragments that are difficult to separate from light membranes by conventional differential centrifugation. We have addressed this problem in two direct ways. One was to measure the presence of Chl throughout the isolation procedure and the other was to label chloroplasts (isolated from lysed protoplasts) in the same fashion as the protoplasts and follow the label as previously described. Table III indicates that all the Chl was recovered in the 10,000g fraction and this was substantiated by the lack of perceptible Chl contamination of the final 80,000g pellet. In addition, 95% of the label was recovered in the initial 10,000g pellet and 6% from the supernatant of the first 80,000g centrifugation (Table III), this latter 6% possibly being accounted for by light chloroplast envelope membranes or soluble protein.

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analysis on sucrose density gradients indicated that the membranes were separate and distinct from major organelles (Fig. 4). In addition, the position of these membranes on the sucrose density gradient agrees with the cell surface labeling study of Galbraith and Northcote (5). This confirms other work in establishing the position of plasma membranes in sucrose density gradients (9, 10,

14-16). Partial characterization of the plasma membrane ATPase indicated that the activity was Mg2"-dependent, K+-stimulated (Table IV), and showed a pH optimum near neutrality (Fig. 3). In addition, the enzyme is substrate specific, showing a distinct preference for ATP over other substrates (unpublished data). This enzyme activity is characteristic of root and coleoptile plasma membranes (6, 8, 9, 13-15) and has also been detected in leaf tissue (16). While a K+-stimulated ATPase was readily detected, Table II indicates that this enzyme is best utilized as a qualitative marker rather than a quantitative marker. This is due to the presence of nonspecific phosphatases which are present in large amounts. The K+-stimulated ATPase activity was reflective of the plasma membrane fraction isolated and did not represent contamination due to mitochondria since ATPase activity was unaffected by the presence of oligomycin (Fig. 5). Protoplasts proved to be effective for cell surface isolation since internal organelles could be maintained intact and thereby be rapidly separated from lighter membranes. However, due to the gentle nature of the lysis process, plasma membrane yields tended to be less than optimal. This was readily seen by determining the total label recovered in the 80,000g pellet (fraction D), an amount which was always less than 50%o (Table I). This loss was probably due to the predominant formation of large, dense membrane 8-5 7-5 5.5 6-5 4.5 sheets during lysis as opposed to small, light vesicles. The large pH sheets were readily moved and pelleted under relatively low FIG. 5. A profile of ATPase activity as a function of pH in the presence centrifugal fields (lO,000g). and absence of oligomycin. (A) control membranes (0) and control We conclude that a plasma membrane-enriched fraction may membranes incubated with 50 ,ug/ml oligomycin (0); (B) control mem- be isolated from corn leaf mesophyll protoplasts by gentle lysis, branes supplemented with mitochondria (0), and supplemented mem- and differential and density gradient centrifugation. An external branes incubated in the presence of oligomycin (0). label, diazotized ['251]iodosulfanilic acid, was used to monitor the cell surface membranes and a correlation was established between DISCUSSION label and enzyme activity associated with the label. Analysis for organelle contamination indicated that the plasma membranes A plasma membrane-enriched fraction has been isolated from were of high purity.

leaf mesophyll protoplasts. It has been positively identified by labeling with diazotized ['25I]iodosulfanilic acid. External labeling of the plasma membrane is the most effective means of identifying this subcellular membrane when intrinsic markers are absent or in doubt. The basic requirements used to select an effective cell surface label have been outlined by Maddy (17). It should (a) be nonpermeant and small for accessibility; (b) react under physiological conditions of pH and temperature; (c) form stable, covalent linkages with membrane constituents; (d) be detectable in small amounts; and (e) cause little or no perturbation of membrane structure and function. Diazotized [1 Iliodosulfanilic acid was chosen because it readily met all of the above criteria and has been shown to be an effective label in other systems (4). The labeling of purified protoplasts with this external marker permitted rapid identification of the plasma membrane during fractionation. Table I demonstrates that label was detected in each fraction but the 80,000g pellet (fraction D) was markedly enriched in label. It is reasonable to suggest that this fraction was highly corn

enriched with cell surface membranes. A previous labeling study has demonstrated the isolation of a highly labeled plasma membrane-enriched fraction. However, those membranes displayed significant golgi contamination (5). In contrast, analysis of fraction D for major organelle contamination indicated that it was relatively free of mitochondria, ER, golgi (Table II), and chloroplasts (Table III). Thus, highly purified plasma membranes must be present in this fraction. Further

LITERATURE CITED 1. AMES BN 1966 Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8: 115-118 2. CASSAGNE C, R LESSIRE, JP CARDE 1976 Plasmalemma enriched fraction from leek epidermal cell. Plant Sci Lett 7: 127-135 3. EARLE ED, VE GRACEN, OC YODER, KP GEMMILL 1978 Cytoplasmic-specific effects of Helminthosporium maydis race T toxin on survival of corn mesophyll protoplasts. Plant Physiol 61: 420-424 4. EDWARDS RM, SA KEMPSON, GL CARLSON, TP DouSA 1979 Diazotized 112511diiodosulfanilic acid as a label for cell surface membranes. Studies on erythrocytes. Biochim Biophys Acta 553: 54-65 5. GALBRAITH DW, DH NORTHCOTE 1977 The isolation of plasma membrane from protoplasts of soybean suspension culture. J Cell Sci 24: 295-310 6. HENDRIX DL, RM KENNEDY 1977 Adenosine triphosphatase from soybean callus and root cells. Plant Physiol 59: 264-267 7. HENDRIKS T 1976 lodination of maize coleoptiles: A possible method for identifying plant plasma membranes. Plant Sci Lett 7: 347-357 8. HENDRIKS T 1977 Multiple location of K-ATPase in maize coleoptiles. Plant Sci Lett 9: 351-363 9. HoDGEs TK, RT LEONARD, CE BRACKER, TW KEENAN 1972 Purification of an ion-stimulated adenosine triphosphatase from plant roots: associated with plasma membranes. Proc Nat Acad Sci USA 69: 3307-3311 10. HODxEs TK, RT LEONARD 1974 Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol 32: 392-406 11. KANAI R, GE EDWARDS 1973 Purification of enzymatically isolated mesophyll protoplasts from C3, C4 and Crassulacean acid metabolism plants using an aqueous dextran-polyethylene glycol two-phase system. Plant Physiol 52: 484490 12. LEMBI CA, DJ MORRE 1971 N- I-Naphthylphthalamic acid-binding activity of a plasma membrane-rich fraction from maize coleoptiles. Planta 99: 37-45 13. LEONARD RT, TK HODGES 1973 Characterization of plasma membrane-associ-

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PLASMA MEMBRANE LABELING AND ISOLATION

ated adenosine triphosphatase activity of oat roots. Plant Physiol 52: 6-12 14. LEONARD RT, CW HOTCHKISS 1976 Cation-stimulated adenosine triphosphatase activity and cation transport in corn roots. Plant Physiol 58: 331-335 15. LEONARD RT, WJ VANDERWOUDE 1976 Isolation of plasma membranes from corn roots by sucrose density centrifugation. An anomalous effect of Ficoll. Plant Physiol 57: 105-114 16. LURIE S, DL HENDRIX 1979 Differential ion stimulation of plasmalemma adenosine triphosphatases from leaf epidermis and mesophyll of Nicotiana rustica L. Plant Physiol 63: 936-939 17. MADDY AH 1964 A fluorescent label for the outer components of the plasma membrane. Biochim Biophys Acta 88: 390-399 18. MARKELL MAK, SM HAAS, LL BIEBER, NE TOLBERT 1978 A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206-210 19. QUAIL PH 1979 Plant cell fractionation. Annu Rev Plant Physiol 30: 425-484

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20. RAY PM, TL SHININGER, MM RAY 1969 Isolation of ,B-glucan synthetase particles from plant cells and identification with golgi membranes. Proc Nat Acad Sci USA 60:605-612 21. STROBEL GA, WM HESS 1974 Evidence for the presence of the toxin-binding protein on the plasma membrane of sugarcane cells. Proc Nat Acad Sci USA 71: 1413-1417 22. TAYLOR ARD, JL HALL 1978 An ultrastructural comparison of lanthanum and silicotungstic acid/chromic acid (STAC) plasma membrane stains of isolated protoplasts. Plant Sci Lett 14: 139-144 23. THOM W, WM LAETSCH, A MARETZKI 1975 Isolation of membranes from sugarcane cell suspensions: evidence for a plasma membrane enriched fraction. Plant Sci Lett 5: 245-253 24. VANDERWOUDE WJ, CA LEMBI, DJ MORRE, JI KINDINGER, L ORDIN 1974 Glucan synthetase of plasma membrane and golgi apparatus from onion stem. Plant Physiol 54: 333-340