Báo cáo khoa học: Localization of fluorescence-labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum pot

Localization of fluorescence-labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum Miachael Karl 1 , Bernd Gasselmaier 1 , Rene ´ C. Krieg 2 and Eggehard Holler 1 1 Institut fu ¨ r Biophysik und Physikalische Biochemie and 2 Institut fu ¨ r Pathologie der Universita ¨ t Regensburg, Germany The nuclei in the plasmodium of Physarum polycephalum,as of other myxomycetes, contain high amounts of polymalate, which has been proposed to function as a scaffold for the carriage and storage of several DNA-binding proteins [Angerer, B. and Holler, E. (1995) Biochemistry 34, 14741– 14751]. By delivering fluorescence-labeled polymalate into a growing plasmodium by injection, we observed microscopic staining of nuclei in agreement with the proposed function. The fluorescence intensity was highest during the recon- struction phase of the nuclei. To examine whether the delivery was under the control of polymalatase or related proteins [Karl, M. & Holler, E. (1998) Eur. J. Biochem. 251, 405–412], the cellular distribution of these proteins was also examined by staining with antibodies against polymalatase. Double-stained plasmodia revealed a fluorescent halo around each fluorescent nucleus during the reconsititution. Fluorescent nuclei were not observed when the hydroxyl terminus of polymalate, known to be essential for the binding of polymalatase, was blocked by labeling with fluorescein-5-isothiocyanate. By immune precipitation, it was shown that polymalate and polymalatase or related proteins were in the precipitate. It is concluded that polymalate is delivered to the surface of nuclei in the com- plex with polymalatase or related proteins. The complex dissociates, and polymalate translocates into the nucleus, while polymalatase or related proteins remain at the surface. Keywords: Physarum polycephalum; plasmodium; polymalic acid; polymalatase; reconstituting nuclei. Physarum polycephalum is a well characterized member of the plasmodial slime molds (myxomycetes) that typically have a life cycle involving haploid (spores, amoebae) and diploid (plasmodia) cell forms [1]. Together with the cellular slime mold Dictyostelium discoideum and other Mycetozoa, P. polycephalum has been placed among the multicellular eukaryotes on the basis of molecular phylogenetic criteria [2]. The plasmodium undergoes mitosis without cytokinesis and usually develops into a multinuclear giant cell (macro- plasmodium). This can contain billions of nuclei (e.g. 10 9 nuclei for a cell having a diameter of 14 cm), which divide synchronously within 3–4 min in an 8–9 h cycle. P. poly- cephalum, especially the plasmodium, has been chosen as a model to study biological and biochemical questions concerning differentiation and cell cycle [3,4]. Considering the synchronous timing of cellular events and the giant dimension of a plasmodium, an appropriate device is necessary to achieve at any given moment an even distribution of molecular constituents. A characteristic oscillating protoplasma streaming [1] probably accounts for the travelling of material along the veins over a certain distance, although the mechanistic details are not known. To achieve a coordinated delivery of equilocally functioning proteins, molecular vehicles would have the advantage of stockpiling and carrying a number of different molecules jointly to defined loci, for example, nuclear proteins involved in DNA replication to nuclei. Previously, we have isolated a polyanion which could function in this regard ([5,6] and references therein). Poly(b- L -malate) is synthesized only in plasmodia and not in the other cell types of the life cycle. It is found at a constant high level in the nuclei and at lower, variable concentrations in the cytoplasm and the culture medium. The polyanion consists of units of L -malate, which are esterified between the hydroxyl group and the b-carboxyl group to form a linear polyester of a number-averaged molecular mass of 50 000. It binds replicative DNA poly- merases, histones and other nuclear proteins reversibly [5,7– 10]. Polymalate synthetase has been studied in vivo [11], but neither the locus nor the proteins involved in the synthesis could be identified. It is assumed that polymalate transfers through the cytoplasm, becomes loaded with cargo proteins, and delivers them to the nucleoplasm. When exceeding a certain cellular concentration threshold, polymalate is exported to the culture medium and subsequently cleaved to L -malate by polymalatase [12]. The cleavage activity is maximal at pH 4.0. Although large amounts of the enzyme and of other immunologically related proteins are found in the cytoplasm, polymalate is not degraded due to the unfavorable intracellular pH of 6.5 [13]. The high cytoplas- mic abundance, the increased affinity to bind polymalate yet at the same time a decreased hydrolytic activity at pH 6.5, and the mode of binding to the HO-terminus of polymalate Correspondence to E. Holler, Institut fu ¨ r Biophysik und Physikalische Biochemie der Universita ¨ t Regensburg, Universita ¨ tstrasse 31, Regensburg, Germany. Fax: +49 941 943 2813, Tel.: +49 941 943 3030 E-mail: Eggehard.Holler@biologie.uni-regensburg.de Abbreviations: FITC, fluorescein-5-isothiocyanate; DAPI, 4,6-diamino-2-phenylindol. Enzyme: malate dehydrogenase (EC 1.1.1.37). (Received 21 October 2002, revised 7 February 2003, accepted 12 February 2003) Eur. J. Biochem. 270, 1536–1542 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03521.x suggested other additional activities of polymalatase, espe- cially the function of an adapter binding to polymalate and targeting it to nuclei [14,15]. Although polymalate has been proposed to function as a mobile scaffold for the carriage of nuclear proteins, its delivery to nuclei has not been demonstrated. We show here that the polymer, which has been conjugated to fluorescent dye, is quite rapidly transferred into nuclei after injection into the cytoplasm. Moreover, the immunochemical detec- tion of polymalatase or related proteins at the nuclear surface is in agreement with the proposed adapter function. Materials and methods Materials Physarum polycephalum, yellow strain M3CVII ATCC 204388 (high polymalate producer) (American Type Culture Collection) or white strain LU 897 · LU 898 (low poly- malate producer) (R. Anderson, Sheffield), was grown as microplasmodia [16] or macroplasmodia (single cell) [17]. Polymalate was purified from the culture broth of micro- plasmodia and had a number-averaged molecular mass of 50 000 (polydispersity of 2.0) [18]. The covalent coupling of Rhodamine-B-amine (Sigma) via amide formation to polymalate present in a 25-fold molar excess was achieved in water over 4 h at 27 °C (10 m M sodium phosphate buffer, pH 7.5) applying an equimolar amount (dye as reference) of the water-soluble N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide [19]. The dye–polymalate conjugate was precipitated with 75% (v/v) ethanol in the presence of 0.27 M KCl and was further purified on Sephadex G25, eluting in the break-through. In this conjugate, less than 1% of the malyl moieties carried the fluorophor. The terminal hydroxyl group was coupled to fluorescein-5-isothiocyanate (FITC) in the presence of triethylamine following routine techniques [20,21]. The terminal carboxyl group was conjugated to 1-napthyl-ethylenediamine as described previously [22]. The wavelength for excitation/emission were (in nm wavelength): 522/555, 495/525, 320/432 for the conjugate of Rhodamine-B-amine,FITC,N-(1-naph- thyl)ethylenediamine. The number-averaged molecular masses of the conjugates were 11 000, 7000, 17 000 by size-exclusion HPLC with polystyrene sulfonates as stand- ards. Protein A/G (UltraLink TM Immobilized) was pur- chased from Pierce. Protease inhibitor tablets (Complete, Roche Diagnostics, inhibiting serine, cysteine and metallo proteases as well as calpains) and mitochondrial malate dehydrogenase (EC 1.1.1.37) of pig heart were from Roche Diagnostics. Chromatographically purified rabbit anti- serum against polymalatase was the same as used previ- ously [15]. The rabbit polyclonal antibody against BSA was purchased from Sigma (B1520). All other reagents were purchased from Merck (Germany), Pierce (USA) and Sigma (USA) and were of the highest available grade. Methods Preparation of extracts from plasmodia. Microplasmodia, grown for 2 days after inoculation, were collected on a nylon sieve and washed briefly with distilled water. Excess water was removed by spreading on a paper towel. An amount of 1 g was suspended in 10 mL homogenisation puffer containing 15 m M Tris/HCl (pH 7.5), 0.5 m M CaCl 2 , 15 m M MgCl 2 ,5m M EGTA, 500 m M hexylenglycol, 10% (v/v) glycerol, 14 m M 2-mercaptoethanol, and complete protease inhibitor cocktail. The cells were broken by 6–8 strokes in a Dounce homogenator, and pelleted at 4 °Cfor 5 min at 2000 g in a Heraeus Biofuge R17. The supernatant was pelleted again for 15 min at 18 000 g, yielding the cytoplasmic fraction. Immunoprecipitation and hydrolysis of polymalate. One ml of the extracted cytoplasm and 10 lL of rabbit antiserum (20 lg) against p97 polymalatase or against BSA (50 lg for control) were incubated with gentle agitation at 4 °C for 2 h, before being coincubated for 2 h at room temperature with 100 lL settled bed of Ultra- Link TM Immobilized Protein-A/G (previously washed twice with 20 m M sodium buffer, pH 7.5, containing 0.5 M NaCl and twice with 20 m M sodium phosphate buffer, pH 7.5). After centrifugation at 2500 g for 3 min, the pellet was washed three times, each with 500 lL of cell homogenation buffer, and once with distilled water. Aliquots of the pellet were either examined by SDS polyacrylamide gel electro- phoresis or assayed for polymalate. In this case, 20 lLof settled bed were washed three times with 40 lL distilled water and eluted with 40 lL1 M NaCl in water. The eluate was incubated overnight at 37 °C with 120 lLof2 M H 2 SO 4 to hydrolyze polymalic acid to L -malic acid. After careful neutralization with 5 M NaOH, L -malate was assayed fluorimetrically. If slices of polyacrylamide gels containing polymalate had to be assayed fluorimetrically, extraction/hydrolysis was carried out in the presence of 2 M NaOH followed by neutralization with concentrated HCl. L -Malic acid was assayed either photometrically or fluori- metrically [34]. Polyacrylamide gel electrophoresis. Denaturing 7.5% SDS/PAGE was carried out as described by Laemmli [23]. Polyacrylamide gradient (3–10%) gel electrophoresis under nondenaturing conditions was performed as des- cribed by Holler et al. [24]. Silver staining of proteins was according to Heukeshoven and Dernick [25] and Western blotting as described by Towbin et al. [26], employing anti-polymalatase Ig (diluted 5000-fold) as the primary antibody, and peroxidase-coupled anti-(rabbit IgG) IgG as the secondary antibody for staining with 3,3¢-diamino- benzidinetetrahydrochloride as substrate according to Adams [27]. Preparation of plasmodia for fluorescence microscopy. Naturally grown plasmodia were too thick for image processing. To obtain satisfactory optical resolution of structures by fluorescence microscopy, ultra-thin macro- plasmodia were grown for 14–17 h at 27 °C in the dark before staining and fixation, according to the method of Naib-Majani et al. [28]. In control experiments, the progress of the mitotic cycle was followed by the phase contrast technique of Mohberg [29]. For staining, the ultra-thin plasmodia were overlayed with culture medium containing 10 mgÆmL )1 of fluorescence-labeled polymalate or fluores- cent dye alone (control) at the times 10, 30 and 40 min (in S Ó FEBS 2003 Polymalate locates to nuclei of Physarum plasmodia (Eur. J. Biochem. 270) 1537 phase), and 200 min (in G 2 phase) after mitosis. Injection of the fluorescent polymer into the ultra-thin plasmodia was not possible. An efficient transfer from overlaid liquid into plasmodia has been reported for histones [4,30]. Naturally grown plasmodia, routinely of mass 300 mg, were injected with 2 lL of solution containing 10–100 lg fluorescent polymalate or dye alone, as described previously [11]. For fixation, the plasmodia were washed three times with phosphate-buffered saline (1.5 m M KH 2 PO 4 ,8.1m M Na 2 HPO 4 , 140 m M NaCl, 2.7 m M KCl, pH 7.4), and immersed for 1–2 min in ice-cold ethanol or for 10 min in 3% paraformaldehyde. After washing three times in phosphate-buffered saline and once with 10% (w/v) BSA, followed by phosphate-buffered saline for 1 h, the fixed plasmodium was stained with anti-polymalatase Ig (diluted 250–500-fold) for 1 h at 24 °C. This was then washed once with a 1% (w/v) solution of BSA, once with phosphate- buffered saline, and then incubated with FITC-labeled anti- rabbit IgG (diluted 80-fold) as the secondary antibody. DNA was stained with 4,6-diamino-2-phenylindol (DAPI, 0.2 lgÆmL )1 in a solution containing 17.5 gÆL )1 NaCl and 8.82 gÆL )1 sodium citrate). To control the cellular integrity of treated plasmodia, representative samples were stained with tetramethylrhodamin B-labeled phalloidin to demon- strate F-actin bundles. For visualization under the fluores- cence microscope, samples were washed five times with phosphate-buffered saline and embedded in phosphate- buffered saline/glycerol (1 : 1, v/v). Fluorescence microscopy For image processing, the preparations of ultra-thin plasmodia were scanned as optical sections employing a conventional microscope (Axiovert S100, Zeiss, Germany) equipped with an epifluorescence adapter, a Plan-Apochro- mat 63·, 1.40 NA oil immersion objective lens, no additional magnification in front of the camera, a piezoelectric z-axis focus device (resolution 10 nm) and computer-controlled excitation light shutter. For optimal visualization, band pass filters were used as follows: excitation at 480 ± 15 nm (FITC-conjugate, Fig. 1A), 560 ± 15 nm (Rhodamine- B-amine conjugate, Fig. 1B), and 360 ± 20 nm (DAPI, Fig. 1D); emission at 535 ± 15 nm (FITC-conju- gate, Fig. 1A), 630 ± 20 nm (Rhodamine-B–amine conju- gate, Fig. 1B), and 460 ± 25 nm (DAPI, Fig. 1D). Images were recorded with a high resolution (4096 levels of gray, 1317 · 1035 pixels) and peltier element cooled ()15 °C), charge coupled (CCD) camera (Princeton Instruments) employing METAMORPH software (Universal Imaging Corp.). The light haze contributed by fluorescently labeled structures located above and below the plane of optimal focus was mathematically reassigned to its proper places of origin ( EXHAUSTIVE PHOTON REASSIGMENT software from Scanalytics, Billerica, Massachusetts) after accurate charac- terization of the blurring function of the optical system. The blurring function of the optical system was characterized by imaging a through-focus series of optical sections of a 0.22 lm-diameter fluorescent bead (Molecular Probes, Leiden, the Netherlands) using the same optical conditions as those used to obtain the specimen image. Each color was separately recorded and processed, and at the end, images were superimposed. Results All of the dye–polymalate conjugates could be delivered into the plasmodia by injection or the overlay technique. Only nuclei were stained, while other organelles remained dark. The staining intensity depended on the amount of the probes injected, the phase of the cell cycle, and the position of the covalent linkage of the dye to the polymer. Results were indistinguishable for the low (white) and the high (yellow) polymalate-producing strains. Controls with the free dyes replacing the polymalate–dye conjugates did not show the staining. Figure 1B,C,F shows an ultra-thin plasmodium stained by the overlay technique with Rhodamine-B-amine– polymalate in early S phase. The red fluorescence indicates polymalate–dye, the blue indicates chromatin and the green shows polymalatase epitopes. The red fluorescence appears together with the blue DAPI staining (Fig. 1F) indicating the transfer of the probe to the nuclei. These were the only organelles stained. By injection into normally growing Fig. 1. Immunofluorescence microscopy of an ultra-thin macroplasmo- dium (white strain LU 897 · LU 898) in early S phase. Very similar results were obtained with the yellow strains. The picture at a magnification of 630-fold was taken with a CCD-camera and is computer-processed. The red fluorescence shows the position of Rhodamine-B-amine–polymalate. The living plasmodium was at first exposed to the reagent for 30 min. At 60 ± 20 min after metaphase, the plasmodium was fixed and stained with anti-polymalatase Ig fol- lowed by fluorescein-5-isothiocyanate labeled anti-(rabbit IgG) (green fluorescence). In the last step, the nuclei were stained with DAPI (blue fluorescence). For excitation and emission filters see the Methods section. (A) Fluorescein-5-isothiocyanate-labeled antibody staining polymalatase epitopes (green), (B) Rhodamine-amine-labeled polymalate (red), (C) superposition of A and B. (D) DAPI staining of DNA (blue), (E) superposition of A and D, (F) superposition of A, B and D. The bar indicates 5 lm. 1538 M. Karl et al.(Eur. J. Biochem. 270) Ó FEBS 2003 macroplasmodia, the intensity of staining was probed as a function of 10–100 lg of labeled polymalate. At low concentration, the success of staining depended on the time of injection in the cell cycle. The majority of nuclei (>90%) were stained in the first half of S phase. Only a few appeared in G 2 phase and were referred to as unscheduled nuclei. At the higher concentration, all nuclei were simultaneously stained, and the intensity was independent of the time of injection. Staining by the overlay method as in Fig. 1 or by microinjection of 10 lg or less of the conjugate resulted in the same type of fluorescent nuclei that was typical for early S phase. The red fluorescence was distributed unevenly in patches (Fig. 1F). The fluorescence was arranged around the center of the nucleus, and matched only the peripheral area of the blue DAPI fluorescence of chromatin. The patches were reminiscent of the patchwork-like structure of reconstituting nuclei in early S phase seen by phase contrast illumination (e.g. [29]). In the case of staining with high amounts of the dye–polymalate conjugate, the nuclei were evenly stained. A very similar result was obtained when the plasmodium was stained with polymalate conjugated to N-(1-naph- thyl)ethylenediamine at the carboxyl terminus (results not shown). However, the staining of nuclei could not be observed for polymalate conjugated to FITC at its hydroxyl terminus. These plasmodia showed an enhanced back- ground fluorescence only. It may be concluded from these results that for successful delivery into the nucleus the terminal hydroxyl group has to be unsubstituted, whereas pending and terminal carboxyl groups could be derivatized. The kinetics of staining were followed after the injection of 100 lg Rhodamine-B-amine–polymalate in G 2 phase by observing nuclei at a distance of 5 cm from the injection point. Fluorescence was detected after 2 min and displayed at maximum intensity after 8 min. While the fluorescence gradually decreased in the nuclei over the next few hours and finally disappeared after 24 h, it concomitantly appeared in the agar medium. The dye extracted from the agar showed the fluorescence of Rhodamine-B-conjugated– polymalate and the number-averaged molecular mass of 2000. The results suggested that the fluorescent polymalate probe was delivered rapidly to nuclei, thereby traversing large cellular distances. Over a prolonged period of time, the conjugate was secreted into the culture medium, and exocytosis was followed by a trimming of the polymer chain as has been observed previously [12]. Polymalate and polymalatase-like protein(s) migrate jointly to nuclei It has been suggested that polymalate may not be free in the plasmodium but bound to an adapter [15]. Polymalatase has been proposed, because it specifically binds to the hydroxyl- terminus of polymalate in vitro [14] and is devoid of enzyme activity at pH 6.5 in the cytoplasm. We investigated whether the putative adapter was delivered together with the injected Rhodamine-B-amine–polymalate to the nuclei. After the injection of low amounts of polymalate–dye conjugate and fixation, the plasmodium was incubated with antiserum against polymalatase and stained with FITC-labeled secon- dary antibody. Green fluorescent material was found deposited around nuclei, which harbored the red fluorescent Rhodamine-B-amine–polymalate (compare Figs 1A,C,F). The green and the red fluorescence were completely separated from each other (Fig. 1C). The fluorescent material scattered elsewhere in Fig. 1 is thought to belong to pieces of halos not focussed to the plane. The occurrence together of the green halo and the red fluorescence was verified in more than 90% of the fluorescent nuclei. Thus, in early S phase almost all of the nuclei could be stained at low concentrations of the red polymalate–dye conjugate, and these nuclei exhibited the halo of green FITC-labeled polymalatase. Plasmodia which had not received the polymalate–dye conjugate nevertheless showed fluorescent halos, indicating that the conjugated dye was not a stimulus for halo formation. These halo intensities compared with those for polymalate-injected plasmodia, suggesting that the amount of the intrinsic polymalate was not limiting. Halos were absent in G 2 phase except around those very few red fluorescent nuclei that were thought to represent unsched- uled S phase nuclei. The nuclei, which became fluorescent only after injection of high amounts (100 lg) of polyma- late–dye conjugate, did not display the halo. An explanation was that these nuclei incorporated small amounts of polymalate and that the level of deposited halo was in these cases below detection limit. The interior of the nuclei did not show green fluorescence (Fig. 1A), indicating that poly- malatase did not enter nuclei. The results are in support of the simultanous transfer of polymalate and polymalatase to the surface of the nuclei, the dissociation of this complex, and the entrance of polymalate only into the nucleus. Polymalate and polymalatase are constituents of an in vivo complex While a direct in vivo demonstration of the polymalate– polymalatase complex was technically not possible, the following results show support for it. In a first approach, a complex was demonstrated by nondenaturing electropho- resis of the cytoplasmic fraction of the yellow strain on gradient polyacrylamide gels. Immunoblotting revealed polymalatase as a broad band centered at 450 kDa (Fig. 2A). SDS gel electrophoresis of the cut-out band and immunoblotting indicated bands at 220 kDa, 125 kDa, 97 kDa, 68 kDa and 45 kDa as in previous investigations, which were supportive of a precurser, two forms of polymalatase and proteolytic fragments [31]. Analysis of the polymalate content in the cut and eluted nondenaturing gel indicated a peak in the 200–600 kDa range (Fig. 2B). In contrast, a sample of purified polymalate migrated at a position of 100 kDa and below. The addition of spermine hydrochloride to the sample from the cytoplasm caused a shift in the polymalate content of the peak towards positions of lower molecular masses, as was expected on the basis of the dissociative effect of sperminium ions [5,9]. The amount of polymalate in the sample of the cytoplasmic fraction was 1 lg. When 0.5 lg of purified polymalate was added to this sample, the polymer content in the 100–600 kDa position increased marginally, indicating that the free capacity of the protein(s) in the cytoplasm was limited in binding further polymalate. The results are in agreement with the assump- tion of a polymalatase–polymalate complex, but the binding to other proteins cannot be excluded. Ó FEBS 2003 Polymalate locates to nuclei of Physarum plasmodia (Eur. J. Biochem. 270) 1539 In a second approach, polymalate and polymalatase in the cytoplasmic fraction of the yellow strain were copreci- pitated by specific anti-polymalatase Igs immobilized on protein-A/G-Sepharose. Aliquots of 20 lLoftheloaded Sepharose beads were shown by SDS gel electrophoresis and Western blotting to contain polymalatase (data not presented). Another sample (20 lL) was eluted in the presence of 1 M NaCl. Polymalate in the eluate was hydro- lysed and quantitated by the fluorimetric malate dehydro- genase assay. An average amount of 0.069 ± 0.003 lg polymalate (SD, three measurements) was detected com- pared to 0.003 ± 0.003 lg in the control (anti-polymala- tase serum substituted by rabbit antiserum against BSA). Variation of the salt concentration in the precipitation buffer indicated that 0.15 M NaCl sufficed to dissociate polymalate from the Sepharose-bound immune complex. Regarding the balance, polymalate in 1 mL of the cyto- plasmic fraction was 6 lg (100%), the corresponding amount in the precipitate was 0.35 lg (6%). This low amount of coprecipitated polymalate is in agreement with the large excess of polymalate over polymalatase in the cytoplasmic fraction. Discussion Polymalate has been proposed to function as a molecular scaffold that binds nuclear proteins and delivers them to the nuclei of the plasmodium [9]. The details of the in vivo delivery are largely unknown. To investigate the delivery, the polymer was labeled with Rhodamine-B-amine or other fluorescent dyes and microinjected or administered by the overlay technique. The amounts of polymalate probes introduced into a typical macroplasmodium (300 mg) were of the order of 10–100 lg, corresponding to 5–50% of the total polymalate content (200–250 lg [13]). The immediate delivery to the nuclei, indicated by their fluorescence staining, was followed by microscopy, while the amount of the probe and the time of administration relative to the cell cycle were varied. After the administration of small amounts of the probe, high numbers (>90%) of nuclei were stained only in early S phase. The few nuclei in G 2 phase were referred to as lacking synchrony. At large amounts of the probe, nuclei were fluorescent independently of the phase of the cell cycle and were evenly stained. At low probe concentrations, however, the fluorescence intensity was distributed in patches, and these did not coincide with the intensity of the DAPI-stained DNA. In the plasmodium, the early S phase immediately follows mitosis and coincides with the phase of nuclear reconstitution [29]. The fluorescent patchwork could correspond to the existence of the reported higher order complexes of polymalate and nuclear proteins [9,10]. The bright spots are indicative of the reconstituting nucleolus [29], reflecting complexes of polymalate with nucleolus-specific proteins, such as the basic fibrillarin-like protein B-36 [32]. The high degree of staining in the early S phase correlates with the reported massive incorporation of 14 C-polymalate into nuclei during feeding of plasmodia with D -[ 14 C]glucose [13]. Because the request for nuclear Fig. 2. Comigration of polymalatase and polymalate in nondenaturing polyacrylamide gradient (3–10%) gel electrophoresis. (A) Western blot with anti-polymalatase serum. (B) The gel was cut at the positions of molecular masses indicated in the figure; the pieces were extracted and assayed for polymalate. Molecular mass standards were BSA, ferritin and their oligomers. The samples were: 0.5 lg of purified polymalate; a mixture of purified polymalate and 50 nmol spermine hydrochloride; 100 lL cytoplasmic fraction (1 lg polymalate); a mixture of 100 lL cytoplasmic fraction and purified polymalate; a mixture of 100 lL cytoplasmic fraction, purified polymalate, and 50 nmol spermine hydrochloride. The mixtures were incubated for 10 min on ice before electrophoresis. 1540 M. Karl et al.(Eur. J. Biochem. 270) Ó FEBS 2003 proteins is high in early S phase, the observed specific and rapid delivery of polymalate to nuclei in the reconsti- tution phase is in agreement with the proposed carriage function. The necessity for high concentrations of the probe to achieve staining of nuclei in G 2 phase is explained by a decreased rate of polymalate delivery and could reflect the transport of different proteins in G 2 phase compared to early S phase. Polymalate recycles to the cytoplasm for new rounds of delivery. As new molecules of the polymer are continuously synthesized, a fraction of the recycled molecules will be secreted into the culture medium to maintain a constant nuclear level [13]. This is observed as a depletion of the fluorescence from the nuclei and an accumulation of fluorescent polymalate in the culture medium. Polymalatase has been proposed to function as an adapter for polymalate [15] and the binding site has been mapped by inhibitor studies [14]. The hydroxyl-terminus of the polymer is recognized by a structurally rigid subsite on the protein. The chemical substitution of the hydroxyl group by a bulky residue would abolishes the binding for sterical reasons. Indeed, after substitution with fluorescein- 5-isothiocyanate the staining of the nuclei was abolished. This result supported the assumption that the delivery of polymalate to the nuclei required the binding to poly- malatase. Evidence for a polymalate–polymalatase complex was provided by the results of electrophoretic and copre- cipitation experiments. Simultaneous staining observed by polymalate–dye and anti-polymalatase Igs suggested that polymalate and polymalatase comigrated to the nuclei. The assumption of a comigration is corroborated by the fact that polymalate and polymalatase are soluble in the cytoplasm and form a complex with each other. The deposition of polymalatase on the surface of the nuclear envelope is thought to involve the docking of polymalatase to certain envelope proteins that induced the dissociation of the adapter–polymalate complex. While these docking proteins are not known, it is interesting to refer to the observation that the dissociation of the adenovirus Ad2 capsid from virus DNA is mediated by its docking to the nuclear pore complex of the receptor CAN/Nup214 and histone H1 in rat [33]. Alternatively, the dissociation of the polymalate– polymalatase complex could be due to a local ionic strength effect, disrupting the electrostatic interactions between polymalate and the adapter protein [14]. A third possibility could be myristoylation and/or palmitoylation of poly- malatase by a corresponding transacylase located in the outer nuclear membrane, resulting in the binding of the adapter to this membrane and the concomitant release of polymalate. The primary sequence structure of poly- malatase suggests such fatty acid acylations (unpublished data). Whether specific docking proteins, local ionic effects or fatty acid acylation and membrane binding are involved has yet to be clarified. After the dissociation, the released polymalate translo- cates into the nuclei. Translocation through the nuclear pore could involve nuclear location signals of proteins carried by the polymer as cargo (proteins such as histone H1 or DNA polymerases [9]). 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In Biopolymers 3a (Doi,Y.& Steinbu ¨ chel, A., eds), pp. 75–103. Wiley-VCH, Weinheim (Bergstrasse), Germany. 1542 M. Karl et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . Localization of fluorescence-labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum Miachael Karl 1 ,. fluorescein- 5-isothiocyanate the staining of the nuclei was abolished. This result supported the assumption that the delivery of polymalate to the nuclei required the binding to