chloroplast membranes (triazines/photosystem II/electron transport inhibitors/diuron)

Transcription

1 Proc. NatL Acad. Sci. USA Vol. 78, No. 2, pp , February 1981 Botany Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes (triazines/photosystem II/electron transport inhibitors/diuron) KLAus PFISTER*, KATHERINE E. STEINBACKt, GARY GARDNERf, AND CHARLES J. ARNTZENt United States Department of Agriculture/Science and Education Administration/Agricultural Research, Department of Botany, University of Illinois, Urbana, Illinois 6181 Communicated by Anton Lang, October 28, 198 ABSTRACT 2 -Azido- 4 -ethylamino- 6 -isopropylamino-s- triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro4-ethylamino- 6-isopropylamino-s-triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido['4c]atrazine resulted in the covalent attachment of radioactive inhibitor to thylakoid membranes isolated from pea seedlings and from a triazine-susceptible biotype of the weed Amaranthuw hybridus. No covalent binding of azido-atrazine was observed for thylakoid membranes isolated from a naturally occurring triazine-resistant biotype of A. hybridus. Analysis of thylakoid polypeptides from both the susceptible and resistant A. hybridus biotypes by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by fluorography to locate "'C label, demonstrated specific association of the azido["'c]atrazine with polypeptides of the 34- to 32-kilodalton size class in susceptible but not in resistant membranes. Many commercial herbicides inhibit photosynthetic electron transport by interrupting electron flow at the reducing side of photosystem II (PS II) (1, 2). There are several lines of evidence which indicate that this inhibition occurs at the level of a protein-bound plastoquinone called "B" (3). This electron carrier acts as the second stable electron acceptor of PS II (4, 5). It has been proposed that the mode of action of compounds such as atrazine or diuron is via high-affinity binding to the PS II complex (6). Herbicide binding induces a change in the redox potential of the quinone cofactor of B, thus making the transfer of electrons from the primary acceptor (Q) thermodynamically unfavorable (3, 5). In addition, binding may decrease kinetic interactions of Q and B. Evidence that PS II inhibitors interact with a polypeptide of the PS II complex, possibly the apoprotein of B, comes from studies involving the enzyme trypsin (7). Proteolytic digestion of surface-exposed membrane polypeptides results in a destruction of inhibitor binding sites with a concomitant inactivation of the secondary acceptor, B (8). Attempts have.been made to correlate trypsin-mediated changes in polypeptides of the chloroplast membrane or detergent-solubilized PS II particles with changes in inhibitor binding properties (9). To date, however, the polypeptide(s) that determines the inhibitor binding site has not been identified. A variety of radiolabeled inhibitors of PS II have been used for the characterization of the properties of the herbicide-binding site; these studies have resulted in the demonstration of a single binding site per electron transport chain (3, 1). In addition, the affinity of various inhibitors for this site has been determined (6, 8). The association of PS II inhibitors with their The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. 981 binding site is noncovalent. For this reason, attempts at physical isolation of proteins labeled by a radioactive inhibitor have failed because detergent fractionation or electrophoretic separation rapidly leads to a new equilibrium and dissociation of the acceptor-inhibitor complex. The approach we have used to overcome this difficulty in identification of the herbicide receptor is to attach a radiolabeled photoaffinity azido derivative of atrazine to its high-affinity receptor polypeptide in a covalent manner. It is well established that activation of the azido function of photoaffinity-labeled compounds by UV irradiation produces a nitrene that is highly reactive (11). In preliminary investigations this was found to covalently link the azido-atrazine to chloroplast membranes (12, 13). MATERIALS AND METHODS Plant Material. Seedlings of dwarf pea (Pisum sativum Linnaeus 'Progress No. 9,' Ferry Morse Seed, Mountain View, CA) were grown as described (14). Leaves were harvested from 12- to 2-day-old seedlings. Biotypes of Amaranthus hybridus susceptible or resistant to PS II inhibitors were grown in sterile soil. The seed stocks of A. hybridus were derived from plants originally collected in Whatcon County, Washington, and referred to previously as A. retroflexus (15); the correct taxonomic identification for these plants has been provided by L. Wax (Agronomy Dept., Univ. of Illinois, Urbana, IL). Leaves were harvested from 4- to 6-week-old seedlings. Isolation of Stroma-Free Chloroplast Thylakoids. Leaf tissue (2 g) was homogenized in a blender for 5 s at high speed, with 1 ml of cold 1 mm N-[tris(hydroxymethyl)methyl]glycine (Tricine)/NaOH ph 7.8 buffer containing.4 M sorbitol and 1 mm NaCl. Homogenization buffer for A. hybridus leaf tissue contained, in addition, 5 mm Wodium ascorbate and.5% bovine serum albumin. The homogenate was filtered through two and then eight layers of cheesecloth, and the resulting filtrate was centrifuged at Abbreviations: atrazine, 2-chloro-4-ethylamino-6-isopropyl-amino-striazine; azido-atrazine, 2-azido-4-ethylamino-6-isopropylamino-s-triazine; B, a protein-bound quinone serving as secondary electron acceptor for photosystem II; Chl, chlorophyll; CI2indophenol, 2,6-dichloroindophenol; diuron, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea; kdal, kilodalton(s); PS II, photosystem II; Q, plastoquinone serving as primary stable electron acceptor from photosystem II; Tricine, N- [tris(hydroxymethyl)methyl]glycine. * Present address: Institut fur Botanik, Universitat Wurzburg, M. Dallenbergweg 64, -D-87 Wurzburg, Federal Republic of Germany. t To whom reprint requests should be addressed at the present address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI t Present address: Shell Development Co., P.O. Box 4248, Modesto, CA

2 982 Botany: Pfister et al. 1 X g for 1 min. The resulting pellet was resuspended in 4 ml of 1 mm Tricine/NaOH, ph 7.8, containing 1 mm NaCl and recentrifuged at 3 X g for 5 min. The pellet, containing stroma-free chloroplast thylakoids, was resuspended to 1-2 mg of chlorophyll (Chl) per ml in 5 mm Tricine/NaOH ph 7.8 buffer containing 1 mm sorbitol, 5 mm MgCl2, and 1 mm NaCl. Chl concentration was calculated by the equations of Mackinney (16). Assays for chloroplast photochemical activity with 2,6-dichloroindophenol (Cl2indophenol) as the electron acceptor were as described (1). Binding Studies. Chloroplast membranes were diluted to a final concentration of 5 kug of Chl per ml in a 1.-ml volume of the final resuspension buffer (2'C). Various concentrations of 14C-labeled azido-atrazine (specific activity 37.1 MkCi/mg; 1 Ci = 3.7 x 11 becquerels) were then added (.7-7,uM final concentration). After an incubation time of 3 min at room temperature (under dim room light) the chloroplast suspension was centrifuged in an Eppendorf 5415 centrifuge at 12, x g for 3 min. From the supernatant,.8-ml samples were removed and the radioactivity of these samples was determined by liquid scintillation counting. From these measurements, the amounts of free and bound inhibitor were calculated. Competition binding experiments were performed in a manner similar to the binding studies described above. A suspension of chloroplasts (13 ml) (5,g of Chl per ml) was first incubated with either.4 AM ['4C]atrazine (specific activity 27.2,uCi/ mg) or.33,um azido[14c]atrazine (specific activity 37.1 AOCi/ mg). Samples were either UV irradiated or incubated in the dark for 1 min. After this incubation, various amounts of [12C]atrazine (1-7 to 2 x 1-4 M final concentrations) were added in small volumes to each sample. The samples were incubated for 3 min and then centrifuged as described above. All inhibitor stock solutions were made up in methanol; the final methanol concentration in any chloroplast suspension was always below 1%. Unlabeled and [ 4C]atrazine were gifts of H. LeBaron, CIBA-GEIGY Agricultural Chemical Division. Unlabeled and azido[14c]atrazine were synthesized by Shell Development, Modesto, CA. UV irradiation was carried out for 1 min with two Sylvania G15T8 germicidal UV lamps located 1 cm above a 5.8-cm diameter glass petri dish containing 13 ml of chloroplast membrane suspension (5 ug of Chl per ml). Proc. Natl. Acad. Sci. USA 78 (1981) Polyacrylamide Gel Electrophoresis. Analysis of membrane polypeptides by NaDodSOJpolyacrylamide gel electrophoresis was carried out by using the discontinuous buffer system of Laemmli (17). Electrophoresis was performed in a slab gel apparatus (18) on a 12-2% (wt/vol) linear polyacrylamide gradient gel and a 5% stacking gel. Membrane samples (5,mg of Chl) were solubilized in 25,ul of sample buffer containing 195 mm Tris-HCl, ph 6.8, 3% (vol/vol) glycerol, 6% (vol/ vol) 2-mercaptoethanol, 6% (vol/vol) NaDodSO4, for 3 min at room temperature. Samples (7-12,ug of Chl) were applied to sample wells. Electrophoresis and staining of gels for protein were carried out as described (19). Polyacrylamide gels were analyzed for covalent 14C-labeled triazine by x-ray fluorography (2) at -8 C, using Kodak SB- 5 x-ray film. RESULTS AND DISCUSSION Characterization of the Azido-Atrazine as an Inhibitor of PS II Photoreactions. It was previously demonstrated that a variety of 2-azido-4-alkylamino-6-alkylamino-s-triazines block light-induced electron transport dependent upon PS II activity (21). Inhibition of PS II-mediated photoreduction of Cl2indophenol by atrazine and azido-atrazine is shown in Fig. 1 for chloroplast membranes isolated from pea seedlings. The concentrations of inhibitor required for 5% inhibition (Iso concentrations) of this reaction were very similar for the two triazines (. 12 pm for azido-atrazine and. 19,uM for atrazine). The replacement of the chloro group of atrazine by the photolabile azido group does not result in markedly changed inhibitory properties. The modification of fluorescence induction kinetics by azidoatrazine is shown in Fig. 2. The induction curve for chloroplast membranes treated with azido-atrazine was identical to that of atrazine-treated samples. Because the relative area above the induction curve is a measure of the site of electron transport inhibition (see ref. 3), these data establish that the actions of azido-atrazine and atrazine are at the same step in the electron transport chain. q) CD 3._ 1 I II a) 2 a) O.= cq ro. '4- C- : 4-1 N4 V C.) 5 - o AO Time of illumination, s I t a Herbicide concentration, M FIG. 1. Inhibition of Cl2indophenol photoreduction in isolated pea chloroplasts by azido-atrazine (o) and atrazine (o). Rates are expressed as percent of control rate in the absence of added inhibitor. The control rate for these experiments was 4 Aeq/mg of Chl per hr. FIG. 2. Modification of Chl fluorescence induction transients of isolated pea chloroplasts by 1 gm azido-atrazine (----) or atrazine ( ). The stimulation in rate of the fast fluorescence rise by azidoatrazine or atrazine in comparison to control ( ) indicates a block in electron transfer at the reducing side of PS II. The data presented are direct tracings of the plotted data obtained from a digital storage oscilloscope; the transient changes observed for atrazine and azidoatrazine were exactly superimposed in plotted form.

3 Botany: Pfister et al. Evidence for Covalent Binding of Azido-Atrazine to Membranes After UVIrradiation. Binding of atrazine to chloroplast membranes is noncovalent and thus reversible. This can be demonstrated either by washing procedures to remove the inhibitor from its binding site or by competition experiments in which the bound inhibitor is replaced by increasing concentrations of an added second inhibitor (see refs. 6 and 1). Evidence for covalent binding of a photoaffinity inhibitor to its receptor site therefore is: (i) that transfer of membranes with bound inhibitor to herbicide-free solution (membrane washing) does not result in release of bound inhibitor and restoration of electron transport activity, and (ii) that the inhibitor is not replaced by a competitive inhibitor. We have compared the reversibility of electron transport inhibition for atrazine and azido-atrazine in samples of pea thylakoids that were either incubated in the dark or treated with UV (Table 1). In the absence of inhibitor, electron transport was affected by UV treatment as reported (22). A 1-min UV irradiation resulted in a 33% decrease of PS II activity. For experiments with atrazine and azido-atrazine as inhibitors, the herbicide concentration was adjusted to give a similar degree of inhibition (7%). In samples that were not irradiated or that were irradiated in the presence of atrazine, the inhibition could be largely reversed by washing (see Table 1). In contrast, inhibition of samples incubated with azido-atrazine and treated with UV could not be reversed by washing. This irreversible inhibition observed in the UV-treated azido-atrazine samples indicates irreversible attachment of the inhibitor to its receptor site Ḣerbicide Binding and Competition Experiments. The results of binding studies with '4C-labeled azido-atrazine are shown in Fig. 3. These data, plotted in double reciprocal form, allow a calculation of the number of binding sites (1 bound inhibitor per 43 Chl molecules) and of a binding constant (Kb) of 32 nm. Both values are in good agreement with the data Table 1. Measurement of reversible herbicide binding to isolated thylakoids Ratio of Cl2indophenol Time of UV Control rate of reduction rates Inhibitor irradiation, Cl2indophenol (after wash/ added min reduction before wash) None Atrazine, M Azido-atrazine, AM Chloroplast membranes (13-ml samples, 5 jig of Chl per ml) were incubated for 1 min in the dark or under UV irradiation with no additions, 2 1AM atrazine, or 12 pm azido-atrazine. Aliquots were then removed, diluted to 5 ug of Chl per ml in the reaction mixture used to monitor Cl2indophenol photoreduction, and illuminated in a spectrophotometer as described (1). The remaining portion of the 13-ml volume was centrifuged at 3 x g. The resultant pellet was washed twice by suspension and recentrifugation in the resuspension buffer. Aliquots of the washed membranes were then removed and assayed for Cl2indophenol photoreduction. All assay mixtures contained, in 2- ml volumes, 1 1g of Chl, 5 mm sodium phosphate buffer (ph 6.8), 1 mm MgCl2, 1 mm sorbitol, 6,uM Cl2indophenol, 1 pm diphenylcarbazide, 2 mm NH4Cl, and 1 MM gramicidin D. The control rates presented were determined for samples prior to washing; values presented are,umol of Ci2indophenol reduced per mg of Chl per hr. The extent of reversal of herbicide inhibition caused by washing (i.e., release of bound herbicide) is indicated by the ratio of Cl2indophenol reduction rate after washing to that before washing; values greater than 1. indicate herbicide release. bo N 'U Proc. NatL Acad. Sci. USA 78 (1981) 983 Free azido-atrazine,,um FIG. 3. Binding of azidol[4clatrazine to pea chloroplast membranes. Analysis of these data by a double reciprocal plot gave a binding constant (Kb) of 32 nm and a value of 1 inhibitor binding site per 43 Chl molecules. Obtained for atrazine binding in Senecio and Amaranthus chloroplasts (one inhibitor per Chl and a Kb of 4-7 nm) (3, 1). Competition experiments demonstrated that noncovalently bound radiolabeled atrazine could be displaced by nonlabeled Unlabeled atrazine, M FIG. 4. Competition of [Cl4Catrazine with unlabeled atrazine in control pea chloroplast thylakoid membranes Mo and membranes that have been treated with UV for 1 min in the presence of [14C~atrazine ().

4 984 Botany: Pfister et al. - ut2 bo2. E -o._ co Q Unlabeled atrazine, M FIG. 5. Competition of azido['4c]atrazine with unlabeled atrazine in control pea chloroplast thylakoid membranes (o) and membranes that had been treated with UV for 1 min in the presence of azido[14c]atrazine (e). atrazine in dark-treated as well as UV-treated membranes (Fig. 4). The extent to which UV damage affected atrazine binding sites is seen from the decrease in binding of ['4C]atrazine in the absence of unlabeled atrazine. The loss of binding sites corresponded closely with the loss of PS II activity in UV-treated membranes. In either control or UV-irradiated samples, however, unlabeled atrazine caused similar displacement of the [I4C]atrazine. UV irradiation also caused loss of binding sites for azido['4c]atrazine (Fig. 5). Bound radioactive azido-atrazine could be displaced by nonlabeled atrazine in the sample incubated in the dark. This result further establishes that both inhibitors act at the same chloroplast membrane receptor site. In contrast to the sample incubated in the dark, the azido-atrazine was not displaced from its binding site by unlabeled atrazine after 1-min UV irradiation. The specificity of the photoaffinity labeling has been further supported by the observations that atrazine protects the site from the UV-induced nitrene, and that photolysis of nonradioactive azido-atrazine at the binding site reduces subsequent [14C]atrazine binding (12). To test the specificity of azido-atrazine binding to the PS II inhibitor site, we compared inhibitor binding to triazine-resistant and susceptible chloroplasts of A. hybridus biotypes. It is now established that, in the chloroplasts of the resistant biotype, the binding site for triazines is selectively altered so that affinity for these inhibitors is greatly diminished (3, 8, 1). The Table 2. Binding of azido-atrazine to chloroplasts from triazineresistant and triazine-susceptible Amaranthus weed biotypes nmol bound inhibitor Chloroplasts Treatment per mg Chl Resistant Dark <.1 UV <.1 Susceptible Dark 2.3 UV 1.25 Chloroplasts were incubated with.5,um azido-atrazine (5,g of Chl per ml). Proc. NatL Acad. Sci. USA 78 (1981) data presented in Table 2 show that the UV-treated resistant chloroplasts bind only a negligible amount of azido-atrazine, as compared to chloroplasts isolated from the susceptible biotypes. This demonstrates that the azido-atrazine binding occurs only in the susceptible membranes and specifically at the high-affinityminhibitor-binding site of the PS II complex. Analysis of membrane polypeptides from resistant and susceptible Amaranthus chloroplasts by NaDodSOjpolyacrylamide gel electrophoresis is shown in Fig. 6 (Coomassie bluestained polypeptides in lanes A). Analysis of the gel by a fluorographic technique showed no detectable bound radiolabel in samples from either resistant or susceptible chloroplast membranes incubated in the dark with azido['4c]atrazine before electrophoretic analysis (data not shown). In samples that were UV irradiated in the presence of azido[14c]atrazine prior to solubilization in NaDodSO4, only the susceptible membranes showed specific covalent attachment of the azido['4c]atrazine to a polypeptide migrating close to stained polypeptides of kdal (lanes B). Lack of covalent binding to this poly- Susceptible A B ams 32 -*- 4S _-P *w j 5_I Resistant A B FIG. 6. Polyacrylamide slab gel electrophoresis of thylakoid membrane polypeptides from susceptible and resistant biotypes of A.-hybridus, stained for protein (lanes A) and by fluorography (lanes B). Susceptible and resistant membranes were incubated with.5,um azido[(4c]atrazine under UV light for 1 min prior to solubilization with NaDodSO4. Arrows indicate polypeptides of 32, 25, and 16 kilodaltons (kdal). The predominant location of the radiolabel as shown by fluorography is over the 34- to 32-kDal polypeptide size classes.

5 Botany: Pfister et al. peptide in the resistant membranes indicates the loss of the triazine receptor region of this polypeptide. Two polypeptides of 25 and 16 kdal were labeled to a minor extent in the susceptible membranes. The polypeptide at 25 kdal corresponds to the major polypeptide of the light-harvesting pigment-protein complex, which is structurally and functionally associated with PS II. It is improbable that the 25- kdal polypeptide contains the atrazine-binding site because it is absent from chloroplast membranes of a Chl b-less barley mutant (23) and partially developed chloroplasts recovered from pea seedlings grown under intermittent light (24), though both of these chloroplasts retain sensitivity to atrazine and other inhibitors of PS II reactions. We have determined that preparations of purified light-harvesting pigment-protein complex do not bind atrazine (data not shown). The fact that the 25-kDal polypeptide does become slightly labeled in susceptible membranes may indicate its close structural proximity to the highaffinity binding site. The significance of the slight labeling of a 16-kDal polypeptide is not clear. It is possible that this polypeptide is also a component of the PS II complex that is in proximity to the triazinebinding site. Alternatively, it is possible that the 16-kDal polypeptide is structurally related to the 32-kDal component via a membrane-localized processing step; this concept will be discussed in a subsequent manuscript. CONCLUSIONS We have demonstrated that azido-atrazine inhibits PS II photoreactions by binding at the receptor site that confers atrazine susceptibility. UV irradiation resulted in the irreversible binding of azido-atrazine to polypeptides of kdal. The fact that binding did not occur in triazine-resistant chloroplasts is an important control showing specificity of labeling of the protein containing the high-affinity binding site. The identification of 34- to 32-kDal polypeptides as the triazine herbicide receptor is consistet with several other observations. Chloroplasts from a maize mutant lacking the 32-kDal polypeptide are blocked in PS II function (25) and lack binding sites for radioactive atrazine (unpublished observations). A 32- kdal protein is present in isolated PS II particles and is removed by trypsin digestion in parallel with loss of diuron (9) binding sites. It has been demonstrated that triazine resistance in intact plants is maternally inherited in Brassica campestris Linnaeus (26); this was correlated to the maternal inheritance of PS II components that confer atrazine sensitivity (27). Chloroplastdirected synthesis of a precursor polypeptide to a 32-kDal protein has been reported (28). In the latter case, the functional role of the chloroplast gene product has not been determined; Proc. Nati Acad. Sci. USA 78 (1981) 985 however, preliminary evidence indicates that the chloroplastdirected polypeptide and the membrane polypeptide labeled by azido['4c]atrazine are structurally identical. We thank Jan Watson for patient technical assistance. This research was supported, in part, by U.S. Department of Agriculture Binational Agricultural Research and Development Grant US 8-79 and a travel grant to K. P. by the Deutsche Forshungsgemeinschaft. 1. Ashton, F. M. & Crafts, A. S. (1973) in Mode of Action of Herbicides (Wiley, New York), pp Wright, K. & Corbett, J. K. (1979) Z. Naturforsch. Teil C 34, Pfister, K. & Arntzen, C. J. (1979) Z. Naturforsch. Teil C 34, Bouges-Boguet, B. (1973) Biochim. Biophys. Acta 314, Velthuys, B. R. & Amesz, J. (1974) Biochim. Biophys. Acta 333, Tischer, W. & Strotmann, H. (1977) Biochim. Biophys. Acta 46, Renger, G. (1976) Biochim. Biophys. 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