An Actin-Related Protein Inside Pea Chloroplasts

An actin-related protein inside pea chloroplasts

DAVID W. McCURDY and RICHARD E. WILLIAMSON

Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, PO Box 475, Canberra City, ACT 2601, Australia* and Department of Botany, La Trobe University, Melbourne

•Address for correspondence

Summary

A pea chloroplast protein resembles vertebrate existed between enzymically and chemically and algal actins by several chemical and immu- generated fragments of the 41000Mr chloroplast nological criteria. On two-dimensional poly- polypeptide and rabbit muscle actin. The acrylamide gels it migrated with a slightly lower 41000 Mr protein was protected from degradation relative molecular mass (Mr = 41000) and slightly by thermolysin only in preparations of intact, but more basic isoelectric point than rabbit skeletal not ruptured, isolated chloroplasts, indicating muscle actin. A monoclonal antibody to chicken that this protein resides within the outer envelope gizzard actin reacted on immunoblots with rab- membrane of these organelles. It is concluded bit skeletal actin, with Chars actin and with a that a 41000 Mr protein with major similarities to 41000Mr band from pea chloroplasts. Pea and actin occurs inside pea chloroplasts, and that a Chara bands of approximately S8000Mr were 58000Mr protein with some similarities to actin also stained. A DNase I-affLnity column that also probably exists within chloroplasts. bound muscle actin also bound 41000 and Key words: pea chloroplasts, actin-related protein, actin 58000Mr chloroplast poly pep tides. Similarities antibodies, DNase I-affinity chromatography.

Introduction showed some of the properties of actin and myosin (Ohnishi, 1964), and roles in light-induced chloroplast Actin is an abundant protein in most, if not all, animal volume changes were proposed (Packer, 1966). While cells (e.g. see Clarke & Spudich, 1977). It functions in little ultrastructural evidence has emerged for filamen- motility and determines many structural features of tous components in these organelles (Newcomb, 1967; both the 'soluble' cytoplasm and the plasma membrane Borowitzka, 1976; Chaly & Possingham, 1981), actin is (Tilney & Detmers, 1975). In plants, actin genes from not always recognizable by such techniques (Tilney & yeast, soybean and maize have been sequenced (Ng & Detmers, 1975; Maupin-Szamier & Pollard, 1978). We Abelson, 1980; Shah et al. 1983) and actin has been therefore re-examined the question of chloroplast actin purified from yeast and tomatoes (Greer & Schekman, using chemical and immunological methods that have 1982; Vahey et al. 1982). The protein has been successfully identified and localized actin in diverse identified in other plants by its ability to bind myosin species including other plants. subfragments (Palevitz & Hepler, 1975), DNase I (Piperno & Luck, 1979; Greer & Schekman, 1982), Materials and methods actin-specific antibodies (Williamson & Toh, 1979; Gallo et al. 1982) and fluorescent phallotoxins Chemicals (Pesacreta et al. 1982). Most of these studies implicate All reagents were analytical grade or better. [l4C]formal- actin in supporting organelle movements of various dehyde (10— ZOmCimmol"1) was from Amersham; Pharma- sorts in plant cells. lytes, Percoll and cyanogen bromide-activated Sepharose-4B were from Pharmacia. At a time when actin's existence outside muscle was not widely accepted, and criteria for its identification Purified proteins were equivocal, suggestions were made that chloro- Chymotrypsin, trypsin and pancreatic DNase I were from plasts contained actin. Thus, chloroplast fractions Sigma, Staphylococcus aureus V8 protease was from Miles Journal of Cell Science 87, 449-456 (1987) Printed in Great Britain @ The Company of Biologists Limited 1987 449 Laboratories and thermolysin from Boehringer. Rabbit skel- resuspended in 200-300^1 of 2% (v/v) Triton X-100 in etal muscle actin (Pardee & Spudich, 1982) was lyophilized 40 mM-potassium phosphate, pH7-0, together with an equal from G-buffer (2mM-Tris- HC1, pH7-5, 0-2mM-ATP, volume of lysis buffer and solid urea to maintain a concen- 0-2mM-CaCl2, 0-2mM-2-mercaptoethanol) and stored at tration of 9-0 M. When protein spots were to be digested —20°C. Monoclonal anti-actin (IgM) was from Amersham with A'-chlorosuccinimide, Triton X-100 was replaced by (N.3S0). CHAPS.

Plant material Radiolabelling by reductive tnethylation Peas (Pisum sativum cv. Massey Gem) were grown as The procedure of Dottavio-Martin & Ravel (1978) was used, described (Jablonski & Anderson, 1981) and watered daily with the following modifications. Actin in 40 mM-potassium with distilled water before harvesting 13-16 days after phosphate, pH7-0, was labelled for 2-D PAGE with 5 mM- 14 imbibition. [ C]formaldehyde (3-4nmol/Jg~' protein) using NaCNBH3 (added as 6mgml~' in 40mM-potassium phosphate, pH7-0) Isolation of chloroplasts in an approximately 11-fold molar excess over the [HC]for- Chloroplasts were isolated from 100 g of pea shoots following maldehyde. The reaction was terminated after 1 h at room the method of Jablonski & Anderson (1981) and the final temperature by acidifying to 50mM-HCl or, prior to affinity chloroplast pellet (referred to as sorbitol-washed chloro- chromatography, by overnight dialysis at 4°C against modi- plasts) was normally used. When required, further purifi- fied G-buffer (G-buffer plus 1% (v/v) Triton X-100). cation of the washed chloroplasts was by centrifugation Sorbitol-washed chloroplasts were labelled by this method through a 10 ml layer of 40% (v/v) Percoll (Mills & Joy, after solubilization in 0-5 ml of 2% (v/v) Triton X-100 in 1980). Osmotic lysis was by resuspension in 5 ml of ice-cold 40 mM-potassium phosphate, pH7-0. lOmM-thioglycollate. After 15min on ice, the supernatant and pellet fractions recovered from a 30 min centrifugation at Fluorography 30 000^ were lyophilized. Coomassie-stained gels were thoroughly rinsed in distilled Two-dimensional polyaerydamide gel electrophoresis water, soaked for 30 min in 1 M-sodium salicylate, 30% (v/v) methanol and 5 % (v/v) glycerol, dried onto filter paper and (2-D PAGE) exposed to Kodak X-Omat AR film at -70°C. The procedure of O'Farrell (1975) was followed, with these exceptions. Isoelectric focusing (IEF) was in 4% (w/v) Proteolytic and chemical digestion of proteins resolved acrylamide gels (30% acrylamide/0-8 % A',Ar'-methylene- bis-acrylamide stock solution) containing 9 M-urea, 2 % (v/v) by SDS-PAGE 14 Triton X-100 and three ranges of Pharmalytes (pH 25 to 5, 4 Proteins were excised from Coomassie-stained gels. C- to 6-4, and 58 to 8; each diluted 1:48 in the final mixture). labelled proteins were digested enzymically for 60 min in the The protein spots digested with A'-chlorosuccinimide were stacking gel (Cleveland et al. 1977) and the fragments were r from gels where Triton X-100 was replaced with 3-[(3- detected by fluorography. Digestion with A -chlorosuccini- cholamidopropyl)dimethylammonio] 1 -propanesulphonate mide was with unlabelled proteins and by silver staining of (CHAPS; Sigma) (Perdew et al. 1983) and the Pharmalyte the fragments (Williamson et al. 1985). mixture by a single range (4 to 6-5 or 5 to 6; diluted 1:16). Polymerization was initiated by adding 0-025 vol. of DNase I-affinity chromatography O'Umgmr' riboflavin containing 1% (v/v) Nflfl'jV'- Pancreatic DNase I was coupled to cyanogen bromide- tetramethylethylenediamine (Temed) and exposing the tubes activated Sepharose-4B (as described by Pharmacia), equi- to fluorescent light for 2h. The electrode buffers were librated in modified G-buffer and poured into 1-1-5 ml 1 M-NaOH and 1M-H2SO4. The sample overlay solution columns (approximately 5mg DNase I per column). All contained 9 M-urea and a 1:32 total dilution of the appro- steps were at 4CC. After washing the column with modified priate Pharmalytes. pH profiles were measured after S mm G-buffer, 1-ml samples of 14C-labelled proteins were loaded gel segments had been shaken for at least 2h in 0-4 ml and the flow was stopped for 15—30 min to maximize binding. of distilled, degassed water. The techniques for one- The elution sequence was: modified G-buffer; modified G- dimensional sodium dodecyl sulphate-polyacrylamide gel buffer plus 0-75 M-guanidine hydrochloride (Gu-HCl); electrophoresis (SDS-PAGE) and immunoblotting were as modified G-buffer with 3-0M-GU-HC1. Samples (0-5 ml) described by Williamson et al. (1986), except that Ponceau S from each 1-ml fraction were mixed with 9-5 ml PCS (2% (w/v) in 30% (w/v) trichloroacetic acid, TCA) was (Amersham) and counted using a Searle Isocap/300 6868 used to stain total protein transferred to nitrocellulose prior to Liquid Scintillation System. When eluates were to be ana- immunostaining. lysed by SDS-PAGE, the extract was added to the equi- librated gel in a test tube and tumbled for 2h at 4°C before Sample preparation for 2-D PAGE packing into a column. Elution was with 10 column volumes Radiolabelled proteins (see below) were lyophilized and of: modified G-buffer; modified G-buffer containing 0-75 M- dissolved in 200-300/il of 90M-urea, 2% (v/v) Triton X- Gu-HCl plus 0-5 M-sodium acetate (pH6-5) (Lazarides 100, 5% (v/v) 2-mercaptoethanol plus Pharmalytes at 1:16 & Lindberg, 1974); G-buffer (column brought to room total dilution. Samples (30—40^1) on each IEF gel were temperature). Final elution was with 5-6ml of G-buffer covered with 20 ^tl of sample overlay solution. With un- containing 05% (w/v) SDS. The lyophilized product was labelled chloroplast proteins, the chloroplast pellet was reconstituted with sample buffer for SDS-PAGE.

450 D. W. McCurdy and R. E. Williamson Protease treatment of isolated chloroplasts with 0-75 M-Gu • HC1 but was eluted with 3 M-Gu • HC1 Methods for the isolation of pea chloroplasts and their (data not shown). Much less of a 14C-labelled chloro- incubation with thermolysin were performed as described by plast extract was tightly bound to a similar column, Cline et al. (1984) with the following modifications. Chloro- but this fraction was substantially enriched for 58 000 plasts were suspended to 1 mg chlorophyll ml"' in wash and 41000Mr polypeptides (Fig. 3). The retained buffer and either ruptured by sonication (30 s with a MSE 41000iWr polypeptide generated chymotryptic frag- sonicator, half-maximal settings for drive and auto) or left ments resembling those from muscle actin (data not intact. Thermolysin was then added to 0-2mgml~' (final shown). concentration of CaCl2 was 0#75 mM) and incubated on ice for 1 h. Control chloroplasts were treated identically but received no thermolysin. Following termination of the reaction with Partial proteolysis EDTA and repurification of the intact chloroplasts (Cline et al. 1984), protein was precipitated with 10 vol. of cold The 41000 Mr chloroplast polypeptide and muscle acetone, collected by centrifugation at 15 000^ for 20min, actin excised from Coomassie-stained 2-D gels gave and solubilized in sample buffer for SDS-PAGE. similar chymotryptic fragments (Fig. 4A) and both yielded prominent, slowly digesting 33 OOOvV/r tryptic fragments (arrows, Fig. 4B). In contrast, the frag- Results ments generated by V8 protease showed only limited similarities (Fig. 4C). With iV-chlorosuccinimide Comigration cleavage, muscle actin gave two fragments of approxi- Radiolabelling by reductive methylation did not mately 40000Mr whereas the pea protein gave one (Fig. 4D). Allowing for the discrepancies in the Mr change the pi or Mr of muscle actin (Heacock et al. 1982) and chemically indetectable quantities of labelled values of the parent bands, fragments of approximately muscle actin visualized by fluorography comigrated by 30000A/r from both proteins corresponded well, 2-D PAGE with an excess of unlabelled muscle actin detected by Coomassie staining (data not shown). Chloroplast proteins prepared in the presence of thio- 200 glycollate and resolved by 2-D PAGE showed a promi- 116 nent spot with an MT of 41000 (arrow, Fig. 1A). The pi of this protein was slightly more basic than muscle 97 *- actin (Fig. 1B,C). If thioglycollate was omitted when the extract was prepared, a row of two or more spots 66 %» appeared to the acidic side of the chloroplast protein (Fig. 1C). These were presumably charge hetero- 45 *~ geneity artifacts (O'Farrell, 1975) resulting from a sulphydryl modification, which thioglycollate prevented. Such single charge modifications differ by 29 «* about 0-1 pH unit and muscle actin focused between the parent chloroplast spot and the first charge- modified form. The 41 000Mr protein was present in comparable amounts following additional chloroplast Fig. 1. Separation of proteins by 2-D PAGE. A. A prominent chloroplast protein (arrow) migrates with an M purification using Percoll (data not shown) and was not r of 41 000 and a pi of approximately 5'4. Coomassie a contaminant of the bovine serum albumin (BSA) staining of chloroplast extract prepared with thiogycollate. used in the extraction buffer. B,C. Relevant regions from a comigration experiment in which 14C-labelled chloroplast proteins were mixed with Immunoblotting excess unlabelled muscle actin so that only the latter was A monoclonal antibody to chicken gizzard actin (Lin, detected by staining. B. Coomassie-stained muscle actin. 1981) reacted on immunoblots with muscle actin, a C. Fluorogram showing that muscle actin migrates to a position (arrow) slightly behind the 41 000il/ chloroplast band of slightly greater Mr containing Chara actin r protein, and between it and the first of a pair of charge (Williamson et al. 1985, 1986) and a 41 000Mr band in the pea chloroplast extract (Fig. 2). A 58000A/ band heterogeneity spots generated by omitting thioglycollate r from all stages of the preparation. The pi of muscle actin was also specifically stained in both the pea and Chara is, therefore, less than 0-1 pH unit more acidic than the pea extracts. protein (see the text). The pH range is approximately 6-5 to 4-5 (left to right) and the highly abundant protein DNase I binding (55 000Mr) is the large subunit of the major stromal 14 A minimum of 57 % of the applied C-labelled muscle enzyme, ribulose bisphosphate carboxylase. Alr values are actin remained on a DNase I column after washing given in all figures (XlO~3).

Actin-related protein in pea chloroplasts 451 whereas those below 20000Mr corresponded less well Discussion (Fig. 4D). We have shown that a pea chloroplast preparation Digestion of intact and ruptured chloroplasts with contains a 41000Afr protein that is chemically and thermolysin immunologically related to actin and also identified a

The 41000iWr chloroplast protein was not degraded 58 000Mr protein that shares two properties with actin. when intact chloroplasts were incubated with thermo- We have provided evidence that the 41 000 MT protein lysin (Fig. 5). Only when chloroplasts were ruptured is chloroplastic in origin and is not exposed on the was thermolysin able to degrade the 41 000 MT protein chloroplast surface. The evidence for identity and to an approximately 39000iV/r fragment (Fig. 5). The location will be assessed separately. presence of undegraded 58 000Mr protein in both the intact and ruptured chloroplast fractions indicates that Identification this protein was resistant to proteolytic degradation by There are small differences in both MT and pi between thermolysin. the 41 OOOiVfr chloroplast protein and muscle actin. Consistent with a stromal location, the 41000A/r However, the pi difference was less than the O'lpH protein remained in the supernatant together with the unit difference observed between muscle actin and the large subunit of ribulose bisphosphate carboxylase sequenced actin of Saccharomyces (Greer & Schek- when osmotically shocked chloroplasts were fraction- man, 1982), and small variations in Mr exist between ated by centrifugation and resolved by 2-D PAGE authentic actins (Garrels & Gibson, 1976; Piperno & (data not shown). The pelleted thylakoid fraction Luck, 1979; GA\o etal. 1982; Williamson et al. 1985). lacked detectable 41 000Mr protein. No actin-like fila- Thus, the MT and pi of the chloroplast protein fall ments were seen by electron microscopy of Triton- within the reported range of MT and pi seen for other permeabilized or unpermeabilized pea chloroplasts actins. using negative staining techniques that detected both The monoclonal antibody to chicken gizzard actin exogenous F-actin added to the preparations (data not used in this study is monospecific in various animal shown) and the actin bundles associated with Cham cells (Lin, 1981). On our immunoblots it stained actin chloroplasts (Williamson, 1979). from rabbit skeletal muscle, the Chara band containing

Ponceau S Anti-actin Control

94-

67- •58

43- -41

-36 -36 30-

20-

M M C M C Fig. 2. Immunoblotting of crude extracts of muscle (M), Chara (C) and pea chloroplasts (P) using monoclonal anti-actin (N.350). The lanes stained for total protein by Ponceau S correspond to those subsequently immunostained with anti-actin. Specifically stained bands are at 42000MT in muscle (plus a smaller M, proteolytic fragment of actin; see Pardee & Spudich, 1982), 43 000 and 58 000iV/r in Chara (plus a lower Mr fragment of the 43 000Mr protein) and 41 000 and 58000Afr in peas. The 36000Mr pea protein is stained in the controls, where the anti-actin was omitted.

452 D. W. McCurdy and R. E. Williamson actin (Williamson et al. 1985, 1986) and the 41 000 Mr pea chloroplast protein migrating just in front of muscle actin (Fig. 2). We consider this strong evidence 116- that the pea protein is similar to actin. Extracts from peas and Chora both contain a second band 97- (Mr = 58 000) reacting with the antibody. This protein is receiving further investigation since a soybean pro- 66- tein of similar MT reacts with various monoclonal and -58 polyclonal antibodies to actin (R. C. Hightower, B. G. 45- McLean & R. B. Meagher, personal communication). In addition, 41000 and 58000Mr proteins present in pea chloroplast fractions also bound to a DNase I- affinity column (Fig. 3). As with extracts of animal 29- cells (Lazarides & Lindberg, 1974; Bretscher & Weber, 1980), other proteins from the chloroplast preparation were retained to some degree by the DNase column.

Muscle actin and the 41 000Mr chloroplast protein were also studied by enzymic and chemical digestion. Enzymic digestion of algal proteins that were clearly 12 3 4 actin-related by other criteria produced peptide maps with similarities to those from vertebrate actins but also 14 Fig. 3. Identification of C-labelled chloroplast proteins with definite differences (Piperno & Luck, 1979; Gallo retained by a DNase I-affinity column. The fluorogram is et al. 1982). The 41 000M pea protein and rabbit actin of a 10% acrylamide gel loaded with samples of: lane 1, r the chloroplast extract applied to the column; lane 2, the showed considerable similarities in chymotryptic frag- initial flow-through fraction; lane 3, the fraction eluted ments and a slowly digested 33 000Mr tryptic fragment with SDS from a control column without DNase I; lane 4, was derived from both (Fig. 4A,B). However, the V8 material eluted with SDS from a DNase column that had protease-derived fragments were less similar (Fig. 4C). previously been washed with 0-75 M-Gu • HC1 containing Among known actins, the maize and soybean actin 0-5M-sodium acetate, pHS-6; 41 000 and 5800OMr bands genes (Shah et al. 1983) have diverged furthest from are prominent. The 41 000Mr band migrated slightly ahead rabbit muscle actin so that discrepancies in peptide of muscle actin and gave similar chymotryptic fragments maps are not surprising. In this situation, cleavage at (data not shown). Trp residues with iV-chlorosuccinimide is attractive since all sequenced actins have only four Trp residues B D whose placement should generate fragments of 40, 38, 3 PM PM PM P M 33, 32, 31, 30, 29 and 28xlO Mr. With muscle and -45 Chara actin (Williamson et al. 1985) we resolved the two largest and four (sometimes five) of the six predicted fragments having Mr values of 28000 to 33 000 (Fig. 4D). With the pea chloroplast protein, the -29 fragments in the 30000A/r region appeared similar but we could resolve only one fragment at approximately 40000Afr (Fig. 4D). This could indicate a missing Trp or simply that the two predicted fragments have -20 identical mobility. Actin sequences predict no fragments of MT11 000-27 000, but under our conditions muscle actin frequently gives one and the pea protein gives several fragments in this range. This must

Fig. 4. Enzymic and chemical digests of the 41 000Mr indicate cleavage at residues other than Trp in muscle chloroplast protein (P) and muscle actin (M). Digestion actin. Cleavage at other residues may likewise occur in products were resolved using 15% acrylamide SDS-PAGE the pea protein or there could be an additional (and for and detected on four separate gels by fluorography (A-C) actin, quite unexpected) Trp in its sequence. or silver staining (D). A. Chymotrypsin. B. Trypsin. These properties indicate important similarities be- The prominent, slowly digesting 33 000Mr fragments are denoted by arrowheads. C. V8 protease. D. A'-chloro- tween the 41 000 MT protein from peas and actins from succinimide. Molecular weight standards (approximate rabbit and the alga Chara. Recent evidence that quite positions) and the position of the undigested bands (M and distinct proteins can have important structural (Maruta P) are indicated on the right. et al. 1984) and functional (Bullard et al. 1985)

Actin-related protein in pea chloroplasts 453 similarities to actin argues for caution, however, in by attachment to their surface or aggregation into large identifying the 41 000Mr protein as authentic actin. We filament bundles. In this regard, however, no filaments therefore refer to it as actin-related. were seen by electron microscopy using techniques that detected muscle F-actin added to pea chloroplast Localization extracts and the bundles of filaments associated with The question then arises of the subcellular location of the surface of the chloroplasts of characean algae both the 41 000 Mr pea protein in particular and also the (Williamson, 1979). 58000M protein. Are the proteins accidentally con- r We used thermolysin, a protease that does not taminating the chloroplast pellet, specifically or non- penetrate the outer envelope membrane of isolated, specifically associated with the chloroplast surface, or located inside the organelle? The presence of actin intact pea chloroplasts (Cline et al. 1984), to examine specifically associated with the chloroplast surface has whether the 41 000iV/r actin-related protein is associ- to be considered seriously because of the prevalence of ated with the chloroplast surface. Under our conditions light-directed chloroplast movements within cells and of incubation, the protease had access to the 41 000.A/r the probable involvement of extrachloroplastic actin in protein only if the chloroplasts were ruptured (Fig. 5). generating the force for movement (Haupt, 1982). Intact chloroplasts prevented degradation of the actin- related protein by thermolysin. These results clearly The 41000A/r protein remained prominent when chloroplast preparations made by the sorbitol washing indicate that the 41 000Mr protein resides within at method were further purified by Percoll gradient least the outer envelope membrane. A stromal location centrifugation. Since each method very substantially of this protein is implied by its presence in the reduces contamination by mitochondria and micro- supernatant fraction of osmotically ruptured, centri- bodies (Mills & Joy, 1980; Jablonski & Anderson, fuged chloroplasts (data not shown). The situation 1981), the likelihood of the 41000Mr protein being concerning a possible chloroplastic location of the derived from a contaminating organelle is slight. How- 58 000A/r protein is less clear, due to the inability of the ever, actin might still co-sediment with the chloroplasts protease to degrade the protein in preparations of Ponceau S Anti - actin Control

67 -58 -41 39 -36 — -36 30- «tf

20-

14- + - R I RI Fig. 5. Immunoblot analysis of pea chloroplast proteins following incubation with thermolysin. Isolated chloroplasts were either ruptured (R) by sonication or left intact (I) and incubated either in the presence ( + ) or absence ( —) of thermolysin (0'2mgml~'). Protein was then precipitated with acetone and resolved by SDS-PAGE (12% acrylamide). The panel stained with Ponceau S was subsequently immunostained with anti-actin, and an equivalent panel of chloroplast proteins used for the control incubation. Note that the 41 000Mr protein was degraded to a 39000Mr fragment only when thermolysin was incubated with ruptured chloroplasts. The 36000A/r pea protein, which stains in the controls where the anti-actin was omitted, presumably is also chloroplastic and is degraded by thermolysin.

454 D. W. McCurdy and R. E. Williamson ruptured chloroplasts. However, its consistent pres- CHALY, N. & POSSINGHAM, J. V. (1981). Structure of ence along with the 41 000MT protein in highly purified constricted proplastids in meristematic plant tissues. and intact chloroplast preparations (Fig. 5) argue that Biol. Cell 41, 203-210. this protein is most likely also chloroplastic. CLARKE, M. & SPUDICH, J. A. (1977). Non-muscle A final argument against actin or an actin-related contractile proteins. The role of actin and tnyosin in cell protein occurring on the surface of isolated pea chloro- motility and shape determination. A. Rev. Biochem. 46, plasts is provided by the continued association of the 797-822. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W. & 41000Afr protein with pea chloroplasts that were routinely prepared in media of low ionic strength. Such LAEMMU, U. K. (1977). Peptide mapping by limited media rapidly release actin from the cytoplasmic sur- proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis.J. biol. Chem. 252, 1102-1106. face of Chara chloroplast envelopes (Williamson, 1978; Williamson et al. 1985). CLINE, K., WERNER-WASHBURN, M., ANDREWS, J. & KEEGSTRA, K. (1984). Thermolysin is a suitable protease Our overall conclusion is that pea chloroplasts con- for probing the surface of intact pea chloroplasts. PL tain a 41000Mr protein resembling actin by several Physiol. 75, 675-678. immunological and chemical criteria and a less DOTTAVIO-MARTIN, D. & RAVEL, J. M. (1978). thoroughly studied 58000Afr protein sharing two Radiolabelling of proteins by reductive alkylation with l4 actin-like features with the 41 000Mr protein (binding [ C]formaldehyde and sodium cyanoborohydride. to the monoclonal anti-actin and to DNase I). The Analyt. Biochem. 87, 562-565. proteolysis data comparing intact versus ruptured EGLY, J. M., MIYAMOTO, N. G., MONCOLLIN, V. & chloroplasts clearly place the 41 000Mr protein within CHAMBON, P. (1984). Is actin a transcription initiation the outer envelope membrane of these organelles. This factor for RNA polymerase B? EMBOjf. 3, 2363-2371. is the first report (except for the inconclusive studies GALLO, J.-M., KARSENTI, E., BORNENS, M., DELACOURTE, noted in the Introduction) of an actin-related protein A. & SCHREVEL, J. (1982). Euglenoid movement in inside a membrane-bounded organelle other than the Distigma proteus. II. Presence and localization of an nucleus (Kumar et al. 1984). y-Actin has been local- actin-like protein. Biol. Cell 44, 149-156. ized by immunofluorescence to sites in skeletal muscle GASRELS, J. I. & GIBSON, W. (1976). Identification and where mitochondria occur (Pardo et al. 1983), but it is characterization of multiple forms of actin. Cell 9, not known whether it is inside or at the surface of these 793-805. organelles. The precise identity of the pea protein can GREER, C. & SCHEKMAN, R. (1982). Actin from probably best be pursued through studies of its gene. Saccharomyces cerevisiae. Molec. Cell Biol. 2, Its function is unknown but chloroplasts display shape 1270-1278. HAUPT, W. (1982). Light-mediated movement of changes (Spencer & Unt, 1965; Chaly & Possingham, chloroplasts. A. Rev. PL Physiol. 33, 205-233. 1981) in which actin could participate, while roles in HEACOCK, C. S., BERNSTEIN, B. W., DUHAIMAN, A. S., the maintenance of membrane shape (Tilney & AMORESE, D. A. & BAMBURG, J. R. (1982). In vitro Detmers, 1975), the organization of soluble enzymes labelling of proteins by reductive methylation: (Masters, 1981) and the initiation of transcription application to proteins involved in supramolecular (Egly et al. 1984) have already been suggested for actin structures. J. Cell Biochem. 19, 77-91. in other situations. JABLONSKI, P. P. & ANDERSON, J. W. (1981). Light- dependent reduction of dehydroascorbate by ruptured We thank J. W. Anderson, C. House, H. G. de Couet, U. pea chloroplasts. PL Physiol. 67, 1239-1244. A. Hurley and J. L. Perkin for advice and assistance. D. W. KUMAR, A., RAZIUDDIN, T., FINLAY, H., THOMAS, J. O. McCurdy was supported by a Commonwealth Postgraduate & SZER, W. (1984). Isolation of a minor species of actin Research Award. from the nuclei of Acanlhamoeba castellanii. Biochemistry 23, 6753-6757. References LAZARIDES, E. & LINDBERG, U. (1974). Actin is the naturally occurring inhibitor of deoxyribonuclease I. BOROwrrzKA, M. A. (1976). Some unusual features of the Proc. natn. Acad. Sci. U.SA. 71, 4742-4746. ultrastructure of the chloroplasts of the green algal order LlN, J. J.-C. (1981). Mapping structural proteins of Caulerpales and their development. Protoplasma 89, cultured cells by monoclonal antibodies. Cold Spring 129-147. Harbor Symp. quant. Biol. 46, 769-783. BRETSCHER, A. & WEBER, K. (1980). Villin is a major MARUTA, H., KNOERZER, W., HINSSEN, H. & ISENBERG, protein of the microvillus cytoskeleton which binds both G. (1984). Regulation of actin polymerization by non- G and F actin in a calcium-dependent manner. Cell 20, polymerizable actin-like proteins. Nature, Land. 312, 839-847. 424-427. BULLARD, B., BELL, J., CRAIG, R. & LEONARD, K. (1985). MASTERS, C. J. (1981). Interactions between soluble Arthrin: a new actin-like protein in insect flight muscle. enzymes and subcellular structure. CRC Crit. Rev. J. molec. Biol. 182, 443-454. Biochem. 11, 105-143.

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456 D. W. McCurdy and R. E. Williamson