tomato actin and myosin: contractile proteins from a higher land plant

Cell Motility 2:131-147 (1982)

Tomato Actin and Myosin: Contractile Proteins From a Higher Land Plant

Maryanne Vahey, Margaret Titus, Richard Trautwein, and Stylianos Scordilis

Department of Biological Sciences, The University at Albany, Albany, New York and Department of Biological Sciences, Smith College, Northampton, Massachusetts

This paper describes the initial isolation of actin- and myosin-like proteins from the cytoplasm of the endocarp tissue cells of the fruit of the tomato, Lycopersicon esculentum. Low ionic strength buffers extracted the 42,000 molecular weight tomato actin in the depolymerized form. Tomato actin can be polymerized in 0.1 M KCI, 2 mM MgClz to form 6 nm diameter filaments resembling rabbit skeletal muscle F-actin in their ultrastructure and pattern of decoration with rabbit myosin subfragment-1 (S-I). Tomato F-actin activates the low ionic strength Mg” ATPase of rabbit S-l up to ten-fold. High ionic strength extracts of tomato yield a myosin- like enzyme whose ATPase activity in 0.5 M KCI is maximal in the presence of K+- EDTA and is repressed in the presence of Mg”’. The column-purified enzyme forms a complex with rabbit F-actin, which can be dissociated by Mg2+ ATP. The low ionic strength Mg’’ ATPase of tomato myosin can be activated ten-fold by rabbit actin and up to nineteen-fold by tomato actin. No activation of the tomato myosin by rabbit F-actin occurs in the absence of free calcium ions.

Key words: higher land plant contractile system, actin activation of myosin, S-1 decoration of actin, polymerization of actin, calcium sensitivity of actomyosin interaction

INTRODUCTION

The force for contractile events in a myriad of nonmuscle vertebrate cells is thought to be generated by a mechanism involving proteins that resemble sarcomeric actin and myosin [Pollard and Weihing, 1974; Goldman, Pollard, and Rosenbaum, 1976; Allen and Allen, 1978al. This hypothesis is also an attractive one to account for

Margaret Titus is now at Department of Biology, Brandeis University, Waltham, Massachusetts 02254.

Preliminary reports on part of this work have been presented (Vahey, Scordilis, and Trautwein, 1978; Vahey and Scordilis, 1979; Vahey and Scordilis, 1980).

Address reprint requests to Maryanne Vahey, Department of Anatomy, Albert Einstein College of Medi- cine, 1300 Morris Park Avenue, Bronx, New York 10461.

0271-6585/82/0202-0131$05.00 0 1982 Alan R. Liss, Inc.

132 Vahey et a1

motile phenomena in the plant kingdom, such as transvacuolar streaming [Mahlberg, 19641, rotational streaming in internodal cells of algae [for a review see Allen and Allen, 1978b], rapid cell elongation in pollen tubes [Condeelis, 19741, and chromosome movements and spindle assembly in endosperm cells [Forer and Jackson, 1975; Forer and Jackson, 19761.

The presence of actin in both lower plants and higher land plants has been widely demonstrated by the use of decoration with rabbit myosin subfragment-1 [see Partha- sarathy and Muhlethaler, 19721. In spite of such compelling morphological evidence, no actin has ever been isolated in any purity from a higher land plant. Myosin has been purified from the lower plant, Nitella flexilis [Kato and Tonomura, 19771 and is the only example of an attempt to characterize the biochemical properties of a component of a plant contractile system.

In order for any meaningful progress to be made on the characterization of plant contractile mechanisms in the same detail as the well described vertebrate cells, there is a need to develop a plant system that will yield its contractile elements in sufficient quantity and reasonable purity to facilitate this biochemical analysis. In this paper, we detail the fundamental properties of the actin- and myosin-like proteins from a higher land plant.

We have chosen the endocarp tissue cells of the fruit of Lycopersicon esculentum, the common tomato, for this investigation. These cells reveal rapid rotational streaming in the peripheral cytoplasm and in the transvacuolar strands when viewed in No- marski differential interference contrast microscopy. The availability of the material in large quantities, as well as its ability to be dispersed in buffers of suitable ionic strength for the isolation of actin and myosin, make it a manageable system for the study of plant contractile proteins.

MATERIALS AND METHODS Protein Purification

Like many other cytoplasmic actins [Gordon, Eisenberg, and Korn, 1976; Gordon, Boyer, and Korn, 19771, the bulk of tomato actin apparently exists in the monomeric form, since very low yields of tomato actin are obtained in isolation pro- cedures that depend on high ionic strength extraction or acetone powders. Therefore, an isolation procedure using fresh tissue and low ionic strength buffers was employed to harvest the tomato actin.

Actin was prepared from L. esculentum by reducing the tomato endocarp tissue to a slurry in a mortar and pestle and mixing 400 ml of the slurry with 200 ml of extraction buffer consisting of 0.1 mM CaC12, 0.5 mM ATP, 3 mM imidazole, pH 7.5, and 0.75 mM 2-mercaptoethanol. It is crucial that the pH of the extract be maintained at pH 7.5 by addition of aliquots of a saturated solution of imidazole base. The slurry was homogenized in a Waring blender by three 1-sec pulses and was then centrifuged at 20,OOOg for 30 min and the supernate was fractionated to 60% (NH4),S04 (AS) using a saturated aqueous AS solution. The precipitate was collected by centrifugation at 100,OOOg for 60 min and was resuspended in 15-20ml of extraction buffer and dialyzed against the same solution (three changes) for 24 hr at 4°C. The dialyzate was clarified by centrifugation at 10,OOOg for 10 min and 6 ml of it was applied to a 1.5cm x 65cm C1-Sepharose 4B column (Pharmacia Fine Chemicals, Piscataway, NJ), which was equilibrated and eluted with extraction buffer. The protein was polymerized to F-actin by the addition of KCl to 0.1 M and of MgClz to 2 mM.

Tomato Actin and Myosin 133

Myosin was isolated from the fruit of the tomato by reducing the endocarp tissue to a slurry in a mortar and pestle and then adding an equal volume (500 ml) of extraction buffer consisting of 1 .O M KCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 30 mM Tris hydroxymethyl aminomethane-HC1 (Tris-HCl), pH 7.5, 20 mM dithiothreitol (DTT), 500 Kallikrien inhibitor units (KIU) of Aprotinin per ml (Sigma Chemical Co., St. Louis, MO), and 0.02% phenylmethylsulfoiiylfluoride (PMSF) dis- solved in dimethylsulfoxide (Calbiochem, La Jolla, CA). The hJmogenate was extracted for 1 hr at 4°C with constant stirring and adjustment of the pH to 7.5 by the addition of aliquots of a saturated solution of Tris base. The extract was centrifuged at 10,000g for 30 min and the resulting supernate was diluted to a final concentration of 50 mM KCl by dialysis at 4°C against lOmM Tris-HC1, pH 7.5,5 mM DTT, 2 mM EDTA, and 0.01% PMSF. The pH of the diluted dialyzate was lowered to 6.3 using 0.5 M acetic acid after overnight dialysis. This solution was allowed to stand for 1 hr at 4°C. The precipitate was collected by centrifugation at 10,OOOg for 60 min and the pellet was resuspended in 0.5 M KCl, 1 mM EDTA, 15 mM Tris-HC1, pH 7.5, 5 mM DTT, 0.01% PMSF, and made 10 mM with respect to MgCl2 and Na2ATP. Ammonium sulfate fractionation, using a saturated solution of (NH4)2S04 with 10 mM EDTA at pH 7.0 was carried out on this suspension. The 30-709'0 fraction was centrifuged at 100,OOOg for 60 min at 4°C to collect the myosin, which was then resuspended in and dialyzed overnight against 0.5 M KCl, 1 mM EDTA, 15 mM Tris-HC1, pH 7.5, 2.5 mM DTT, and 0.01 070 PMSF. The dialysate was made 10 mM with respect to MgC12 and Na2ATP and 0.7 ml was applied to a 0.6cm x 100-cm column of C1-Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated in and eluted with 0.5 M KCl, 1 mM EDTA, 15 mM Tris-HC1, pH 7.5, and 2.5 mM DTT. One milliliter fractions were collected every 10 min.

Skeletal muscle myosin was isolated from rabbit back and hind leg muscles according to the procedure of Kielly and Harrington [1960]. Subfragment-1 (S-1) was prepared from the pure myosin by papain digestion as described by Margossian and Lowey 119731. Rabbit muscle actin was prepared from an acetone powder by the procedure of Spudich and Watt [1971].

Protein Analysis Protein concentrations were estimated by the method of Lowry et a1 [1951] using

a cold trichloroacetic acid (TCA) precipitation of the protein to remove any interfering substances such as plant phenolics. Twicecrystallized bovine serum albumin (BSA) was used as a standard. Absorbance at 290 nm was used to detect protein in the column fractions when buffers containing ATP were used. Absorbance at 280 nm was used otherwise.

ATPase Assay Inorganic phosphate liberation was measured by the method of Martin and Doty

as modified by Pollard and Korn [1973]. High ionic strength myosin ATPase activities were assayed in 1.5-ml aliquots with final concentrations of 0.5 M KC1, 10 mM ATP, 10 mM imidazole, pH 7.0, and either 2 mM K'-EDTA, 10 mM CaC12, or 10 mM MgC12. Low ionic strength actin-activated myosin Mg2+ ATPase activities were measured either with column-purified tomato myosin or with rabbit myosin S-l in final concentrations of 20 mM KCl, 3.3 mM MgC12, 0.1 mM CaC12, 3.3 mM ATP, 10 mM imidazole, pH 7.3, and in the presence or absence of a ten-fold excess in mg/ml of am-

134 Vahey et a1

monium sulfate-purified tomato actin, or column-purified tomato actin, or rabbit skeletal muscle F-actin. Incubation was at 37°C for 60 min.

The titration of the low ionic strength actin activated Mg-ATPase of tomato myosin with rabbit F-actin was done under the conditions described above, with a final concentration of 0.1 mM CaC12. Assays were carried out using molar ratios from 0 to 10 mole of actin per 1 mole of tomato myosin. To study the effect of the removal of free calcium ions, tubes were mixed in an identical manner and ethyleneglycolbis (B-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) was added to a final concentration of 1 mM.

The calcium titration of the rabbit actin activation of tomato mysoin was carried out maintaining a constant ratio of 10 moles of rabbit actin to 1 mole of tomato myosin heavy chain. The free calcium ion concentration was varied from pCa 8.3 to 6 by using a Ca2+/EGTA buffer system accounting for MgZ+, Ca2+, ATP, EGTA, and pH. Con- stants for EGTA and ATP, at pH 7.3, were those of Potter and Gergely [1975], and the pCa values were determined by the iterative computer program of Freund and Fleck (personal communication). The pH of the CaZ+/EGTA assay tubes was carefully monitored and was found not to vary.

The calcium titration of the basal low ionic strength Mg" ATPase activity of tomato myosin in the absence of rabbit F-actin was carried out as described above by using the Ca'+/EGTA buffer to vary the PCa from 8.3 to 6.

The percent maximal activation is expressed in terms of the maximal activation of tomato myosin in the presence of rabbit F-actin, taken as 100%.

Controls for all assays containing actin (actin activations and actin binding studies) consisted of determining the background "inherent ATPases" of the particular actin fraction used. Conditions of these assays were identical to the assays containing the rabbit skeletal myosin S-1 or the column purified tomato myosin. The average of three determinations on three different preparations indicate the following specific activities: skeletal F-actin, 0.8 nmole/min/mg; ammonium sulfate tomato actin, 1 .O nmole/min/mg; column-purified tomato actin, 1 .O nmole/min/mg. These values are three orders of magnitude less than those of myosins measured in the activation or binding assays and can be attributed to the minimal pseudo-ATPase exhibited by the G - F transformations on the ends of the actin filaments [Mannherz and Goody, 19761. Nevertheless, these values were subtracted from the specific activities of the myosins in any assay containing actin in order to prevent even the most remote chance of a false-positive due to contribution of ATPase by the actin fraction used.

Qualitative Assays for the Presence of Actin Qualitative viscosities of the protein samples taken from each step of the purifica-

tion procedure were measured at room temperature in a Cannon-Fenske viscometer. Prior to the assay, the samples were dialyzed overnight at 4°C against buffer F (50 mM KCI, 2 mM MgC12, 10 mM Tris-HCI, pH 7.5, and 5 mM DTT). All outflow times were compared to that of buffer F. Samples exhibiting a greater viscosity than that of buffer F were assigned a positive sign ( + a in Table I ) . Development of flow birefringence was determined by stirring of the sample, previously dialyzed into buffer F, between cross- ed polars. These assays were done at room temperature. Examination of samples for the presence of 6 nm diameter filaments is described under Electron Microscopy.

Quantitative Assays for Actin

The ability of samples to activate the low ionic strength Mg2+ ATPase of either

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136 Vahey et al

rabbit or skeletal muscle myosin S-1 or column-purified tomato myosin was assayed for as described under ATPase Assay. Specific viscosities (qsp) were measured as described above, where qsp = sample time - buffer time/buffer time.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Slab gel electrophoresis in 0.1 Vo sodium dodecyl sulfate (SDS), 15% separating gels with 5% acrylamide stacking gels was performed according to the method of Blat- tler et a1 [1972]. The gels were stained with 0.25% Coomassie brilliant blue R in 50% methanol, 10% acetic acid and were destained in the same solution without the dye. Gels were routinely stained for 24 hr in an effort to increase the binding of the dye to the plant proteins since such proteins exhibited unusually weak affinity for the following dyes: Coomassie brilliant blue R, fast green, amido black, etc. Overloaded tube gels used to demonstrate the comigration of tomato and rabbit actin were run in the Fair- banks system [1971].

Electron Microscopy For electron microscopic observation of tomato actin, a cube of endocarp tissue

was reduced to a slurry in a cold mortar and pestle and suspended in a minimum of standard salt solution (SSS) consisting of 0.1 M KCl, 5 mM MgCI2, 0.1 mM DTT, and 6mM sodium phosphate buffer, pH 7.0. The cytoplasmic preparation was treated with rabbit skeletal muscle myosin S-1 in the presence and absence of 10 mM Mg2+ ATP according to the procedure of Palevitz et a1 [1974]. Prior to electron microscopic study, protein samples from the tomato actin purification steps were dialyzed overnight at 4°C against buffer F. Samples were spread on the grid, rinsed with SSS, stained with 2% aqueous uranyl acetate, and examined in an AEI EM6B microscope operated at 60 kV.

RESULTS AND DISCUSSION Tomato Actin

The tomato protein isolated in low ionic strength buffers fulfills the fundamental structural and biochemical properties required of an actin. Tomato actin can be polymerized in the presence of lOOmM KC1 and 2 mM MgC12. These 6-nm diameter tomato F-actin filaments (Fig. 1) are similar in structure to filaments formed by actin from muscle [Harrington, 19791 and nonmuscle cells [Pollard and Weihing, 19741. The column-pure tomato F-actin is unusually labile and, unlike many other actins, decoration with S-1 does not help to stabilize the filaments. Nevertheless, tomato actin filaments from cytoplasmic spreads are very stable and decorate reversibly with rabbit S-1 to form diagnostic arrowheads with a constant periodicity and polarity of 35 nm (Fig. 2). Such arrowheads are dissociated by Mgz+ ATP (Fig. 3).

While all of the tomato protein gels stain anomalously weak in our hands, SDS gels of the major CI-Sepharose 4B column peak (Fig. 4) indicate that the apparent molecular weight of tomato actin is identical to all actins at 42,000 (Fig. 5). In addition, tomato actin comigrates with rabbit actin in SDS-PAGE (Figs. 5 and 6) . The amount of tomato actin that we can isolate by this method accounts for 6% of the total tomato protein fraction (Table I). This 6% figure must be qualified by the fact that additional proteins may be present in this fraction that control actin lability but that do not appear on SDS-PAGE. Levels of actin found in other nonmuscle cells range from 5-15% [Pollard, 19751.


Fig. 1 . The polymerized tomato actin purified to the step immediately preceding gel filtration is very stable and exhibits an ultrastructural morphology similar to rabbit skeletal F-actin with 6 nm beaded filaments. x 148,000; bar = 0.1 pm.

Fig. 2. The formation of arrowhead structures by the treatment of tomato cytoplasmic extracts with rabbit skeletal muscle myosin S-l in SSS and staining with 2% aqueous uranyl acetate. x 94,000; bar = 0.1 pm.

Fig. 3. The dissociation of the arrowhead structures upon the addition of 10 mM Mg” ATP. X 95,000; bar = 0.1 pm.

The most significant property of a presumptive actin is the ability of the protein to interact with myosin. In 20 mM KCl, the Mg2+ ATPase of rabbit myosin S-1 is activated ten-fold by tomato F-actin that has been purified to the ammonium sulfate step (Table 11). Furthermore, under the same conditions of ionic strength, this tomato actin fraction activated the Mg2+ ATPase of column-pure tomato myosin 19-fold. It is note- worthy that the column-pure tomato actin activated rabbit myosin s-1 by a consistently low 2.5-fold. As stated above, we find that this pure actin fraction will polymerize to

138 Vahey et al

Fig. 4. The elution profile from the CI-Sepharose 4B chromatography of the 60% ammonium sulfate protein. The ordinate indicates the protein monitored at The major peak contains the 42,000 M W actin band on 15% SDS gels (A on inset), is polymerizable with salt, and constitutes column-purified tomato ac- t i n (20 pl of I-mg/ml solution was run on the gel).

TABLE 11. Tomalo Aclin Activation of Muscle S-1 and Tomato Myosin ME" ATPase*

Subfragment -1 Subfragment-l + ammonium sulfate

Column-purified tomato myosin Column-purified tomato myosin +

ammonium sulfate purified tomato actin Subfragment - 1 Subfragment-l + column-purified tomato

purified tomato actin

actin

0.017 f 0.008 0.161 f 0.014

0.010 & 0.006 0.188 + 0.038

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19-fold

0.072 & 0.006 0.177 & 0.014 2.5-fold

*Conditions: 20mM KCI, 3.3 mM MgC12, 0.1 mM CaC12, 3.3 mM ATP, lOmM imidazole-HCI, pH 7.3, 0.035 mg/ml of rabbit myosin S-l or 0.03 mg/ml of column-pure tomato myosin and in the presence or absence of 0.32 mg/ml of ammonium sulfate-purified tomato actin or 0.40 mg/ml of column-purified tomato actin. The specific activities are expressed as pmoles inorganic phosphate liberated per min per milligram protein. Values given are the mean & SD, n = 4.

form 6-nm diameter filaments, which are very labile. It is possible that this low level of activation may be due to the inherent lability of the tomato actin after column purification or to the presence of trace contaminating proteins in the tomato actin that reduce its polymerizability.


Fig. 5 . The 7.5% tubegels showing comigration of tomato actin @)wi th rabbit actin (a). Onehundred and fifty microliters of a 1.5-mgIml solution was run on each gel. The dye front is indicated.

Work is in progress to find an explanation for the lability of column-pure tomato actin. Preliminary evidence suggests that a 72,000-dalton tomato protein may function to control the polymerization of the actin component of the tomato contractile system [Vahey et al, 19801. Nevertheless, tomato F-actin with: 1) a molecular weight of 42,000, 2) 6-nm diameter filaments that reversibly decorate with rabbit myosin S-1, and 3) the ability to activate the low ionic strength Mg2+ ATPase of both rabbit S-1 and column- purified tomato myosin is a bonafide higher land plant actin-like protein.

Tomato Myosin

Tomato myosin appears to be composed of a heavy chain of approximately 100,000 molecular weight and two light chains of 16,000 and 14,000 daltons (Fig. 6). It is important to note that neither the fraction applied to the column, nor the first column peak of low specific activity, nor the third column peak of low molecular

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Rf Fig. 6 . SDS polyacrylamide gel electrophoresis of the column-purified tomato proteins on calibrated SDS gels. The inset shows the actual 15% SDSgel of tomato myosin peak 2, while the specific molecular weight of the myosin's component subunits is indicated on a calibration curve of the gel. The arrow indicates the tomato myosin heavy chain and (B) indicates the light chains. The standard proteins are represented by ( 0 ) and the tomato myosin and actin are represented by ( x ). The solid line is determined by calculation of the difference of least squares to draw the best fitting line.

weight polypeptides, exhibits any bands of greater than 100,000 daltons (Figs. 7a, b , c). As in the case of Nitella myosin, the subunit stoichiometry remains to be determined for tomato myosin. While we have made extensive use of potent proteolytic inhibitors, the possibility of a higher molecular weight for tomato myosin cannot be discounted. It will be of interest to explore this possibility as the only other low molecular weight myosin that has been described is the myosin I of Acanthamoeba [Pollard and Korn, 1973; Pollard, 19791. Further studies requiring larger amounts of pure tomato myosin will be directed toward an investigation of the native molecular weight, stoichiometry, and subunit composition of tomato myosin.

Nevertheless, the most significant property of the 130,000 molecular weight ATPase from the tomato is that it exhibits all of the qualities of a myosin enzyme. The ATPase activity of tomato myosin is characteristic of rabbit skeletal muscle myosin, the majority of nonmuscle cell myosins, and Nitella myosin in that in 0.5 M KCI the highest activity occurs in the presence of K+-EDTA. The Ca++-stimulated activity is one fourth the K+-EDTA activity and the Mg"-stimulated activity is one sixth the maximal activity (Table 111). The second peak from the gel filtration column contains the highest


Fig. 7. The elution profile from the CI-Sepharose 4B chromatography of the 30-70% ammonium sulfate fraction of tomato myosin. The void volume and the salt boundary were determined by chromatography of blue dextran and ATP, respectively. Fractions were monitored at Also for protein (-) and assayed for K+-EDTA ATPase activity in 0.5 M KCI (---). The inset shows SDS-PAGE of (a) column load, (b) peak 1, and (c) peak 3. Column-pure myosin of peak 2 is shown in Figure 6 .

K+-EDTA ATPase activity (Fig. 7). In addition, only this peak exhibited a behavior characteristic of myosin in actin activation assays and actin binding studies.

Table IV shows the results of two experiments on column-purified tomato myosin from different preparations, indicating that the myosin binds to rabbit F-actin in the absence and not in the presence of Mg2+ ATP. Furthermore, tomato myosin Mg2+ ATPase in 20 mM KCl is activated up to ten-fold by rabbit actin and nineteen-fold by

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TABLE I V . Binding of Tomato Myosin to F-Actin"

K+-EDTA ATPase specitic activity of supernate

Percent ATPase remaining in supernate _ _ _ ~ _ _ _ _ _ _ _ ~ _ _ -

Experiment-1 Experiment-2 Experiment-l Experiment-2 ~

Supernatant fraction ~~ -

Column myosin + F-actin + ATP 0.064 0 024 74 70 Column mvosin + F-actin 0 024 0.008 26 30

*Column-purified tomato myosin (0.4mg/ml) was suspended in 0.5 M KCI, 15 mM Tris-HCI, pH 7 . 5 , 2.5 mM DTT, 1 mM EDTA, 0.1 mM MgCI2 with either muscle F-actin (0.6 mg/ml)or with F-actin and IOmM ATP. The solutions were incubated at 37°C for 30min and centrifuged at 100,OOOg for 60min at 4°C. The supernates were assayed for ATPase activity in 0.5 M KCI as described in the text. Specific activities are expressed as pmoles inorganic phosphate liberated per min per mg protein.

TABLE V. Actin Activation of Tomato Myosin*

Ma" ATPase soecific activity Mean activation

Tomato myosin 0.007 f 0.003 Tomato myosin + rabbit F-actin 0.081 f 0.030 Tomato myosin 0.010 f 0.006 Tomato myosin + ammonium sulfate 0.188 f 0.038

10-fold

19-fold purified tomato actin

*Conditions: 2OmM KC1, 3.3 mM MgCI2, 0.1 mM CaCI,, 3.3 mM ATP, 10mM imidazole-HCI, pH 7.3, 0.030 mg/ml of column-purified tomato myosin and in the presence or absence of 0.30 mg/ml rabbit skeletal muscle actin or 0.32 mg/ml of ammonium sulfate-purified tomato actin. Specific activities are expressed as pmoles inorganic phosphate liberated per min per mg protein. Values given are the mean * SD, n = 4.

tomato actin (Table V). Since actin activation of the low ionic strength Mg2+ ATPase of myosin is the single most diagnostic feature of the enzyme, we further characterized this reaction.

By varying the concentration of F-actin, we found that tomato myosin is maxi- mally activated at a molar ratio of seven rabbit actin to one tomato myosin heavy chain (Fig. 8). This is remarkably similar to the actual ratio of six actin thin filaments to one muscle myosin thick filament that is found in sarcomeric myosin [Harrington, 19791. If this same experiment is done in the presence of the calcium chelator EGTA, there is a dramatic inhibition of the actin activation (as shown by the broken line in Fig. 8). Furthermore, at this ratio of ten rabbit actin per one tomato myosin heavy chain, the tomato myosin exhibits optimal activation at a pCa between 7.5 and 6 in the presence of the nonregulated actin (actin devoid of troponin and tropomyosin, as assayed on SDS polyacrylamide gel electrophoresis), but shows no calcium sensitivity in the absence of rabbit F-actin (Fig. 9).

There are two possible explanations for the inhibition of the low ionic strength Mg2+ ATPase actin activation of the tomato myosin in the presence of optimal levels of rabbit F-actin upon the removal of free calcium ions. Tomato myosin could be considered a regulatory myosin in that direct binding of calcium ions to the molecule is required to overcome some inherent inhibition of the actin activation, not unlike scallop and molluscan myosins [Szent-Gyorgyi, 19801. The remaining possibility is that a Ca2+- activated kinase, specific for phorphorylation of the tomato myosin molecule, medi- ates the actin activation [Adelstein and Eisenberg, 19801. While the point at which the

144 Vahey et a1

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0 2 4 6 8 10 12 14 16 18 20 22

[ACT14 r)l

Fig. 8. The titration of the actin-activated low ionic strength Mg*+ ATPase of tomato myosin by rabbit skeletal muscle F-actin. The ordinate indicates the percent maximal activation and the abscissa shows the concentration of added actin in pnoles. Theassay was performed in the presence (-)and absence (----)of free calcium ions (0.1 mM CaClz and 0. I mM CaC12 + 1 mM EGTA, respectively). The concentration of tomato myosin was held constant at 2.2 pmoles. Optimal activation occurs at a molar ratio of six rabbit actin on one tomato myosin. 100% activation = 0.0586 rmole Pi/min/mg; n = 4 f SE.

calcium ion is exerting its regulatory effect in our system remains to be determined, the most definitive features of the 130,000 molecular weight myosin like ATPase from the tomato are: 1) reversible binding to rabbit F-actin and 2 ) activation of the low ionic strength Mg2+ ATPase by rabbit F-actin in a calcium-sensitive manner.

In some systems of taxonomic classification, Physarum and Dictyostelium are considered as plants. Tomato myosin, however, appears to be distinct from Physarum myosin, molecular weight 480,000 [Adelman and Taylor, 1969a, b], and Dictyostelium myosin, molecular weight 480,000 [Clarke and Spudich, 19731, in having a lower molecular weight. Tomato myosin resembles rabbit skeletal muscle myosin in having maximal high ionic strength ATPase activity in the presence of K+-EDTA. However, Physarum and Dictyostelium show a maximal ATPase activity in the presence of Ca" [Adelman and Taylor, 1969a, b; Clarke and Spudich, 19731.

This initial isolation and partial characterization of tomato actin and myosin in the in vitro interaction of these proteins to hydrolyze ATP suggests that they interact in a mechanocoupling event analogous to sarcomeric myosin and actin. This is compelling evidence for the theory that the force necessary to support cytoplasmic streaming in higher plants is generated by a mechanism similar to the sliding filament model


C 0 .- c

> .- .- 8 - m x E .-

2 8

100

80

60

40

20

Fig. 9. The titration of the calcium dependence of the low ionic strength Mg" ATPase actin activation of tomato myosin. The ordinate indicates the percent maximal activation and the abscissa shows the free calcium ion concentration in terms of pCa. The assay was done in the presence (-)and the absence (----) of rabbit skeletal muscle F-actin. The assay was done while maintaining a molar ratio of rabbit actin to tomato myosin heavy chain at 1 O : l . 100% activation = 0.0557 fimole P,/min/mg; n = 4 + SE.

that accounts for muscle contraction, as well as for a variety of events in the animal cell cytoplasm. The primary importance of this paper, however, lies in the fact that it is a starting point for the detailed characterization of plant contractile systems and their regulation that is sure to follow.

ACKNOWLEDGMENTS

We are most grateful to Dr . Henry Tedeschi for his support and encouragement during this study and for his help in preparing this manuscript. We would also like to thank Drs. Colin Izzard, Charles Edwards, and James Flynn for many valuable discus- sions. We are grateful to Ryland Loos, Robert Speck, and James Yates for help in preparing the figures. We would also like to thank Dr. Peter Chantler for critically reading the manuscript.

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