A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum

A full-length cDNA coding a calmodulin (CaM)-dependent protein kinase gene was cloned from Physarum plasmodia poly(A)-RNA by polymerase chain reaction with the oligonucleotide primers that were designed after the amino acid sequence of highly conserved regions of myosin light-chain kinase. Sequence analysis of the cDNA revealed that this Physarum kinase was a 42,519-Da protein with an ATP-binding domain, Ser/Thr kinase active site signature, and CaM-binding domain. Expression of the cDNA in Escherichia coli demonstrated that the Physarum kinase in the presence of Ca2+ and CaM phosphorylated the recombinant phosphorylatable light chain (PLc) of Physarum myosin II. The peptide analysis after proteolysis of the phosphorylated PLc indicated that Ser 18 was phosphorylated. The site was confirmed by the failure of phosphorylation of PLc, the Ser 18 of which was replaced by Ala. The physiological role of the kinase will be discussed with special reference to the 55-kDa kinase, which had been previously purified from Physarum plasmodia for phosphorylated PLc.     

Characterization of the nucleolar protein, B-36, using monoclonal antibodies

A panel of nine monoclonal antibodies has been produced against a major nuclear protein, B-36, purified from the slime mold Physarum polycephalum. B-36, a 34 kD protein biochemically similar to the major structural proteins of mammalian hnRNP particles, was previously shown to be largely associated with the nucleolus. Eight of the monoclonal antibodies are specific for B-36 protein in Physarum and at least three different epitopes are represented among these eight. Using the monoclonal antibodies B-36 has been shown to be localized exclusively to the nucleolus in actively-growing Physarum cultures. The nucleolar localization of B-36 is dependent on the presence of intact RNA, but not DNA, supporting the hypothesis that B-36 is associated with nucleolar RNA, possibly in some analogous manner to the interaction of the related proteins within heterogeneous nuclear ribonucleoproteins (hnRNP) particles. B-36 is apparently a highly conserved nucleolar protein in eukaryotes as all eight of the monoclonal antibodies specific for B-36 in Physarum are also specific for a 34.5 kD nucleolar protein in rat liver. This indicates that a minimum of three distinct epitopes are conserved in B-36 protein from slime mold to rat.     

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.     

Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon.

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.     

Ca2+-binding modulator protein in protozoa and myxomycete.

Ca2+-binding protein with the properties of brain modulator protein of 3,5-cyclic nucleotide phosphodiesterase was identified in Physarum polycephalum plasmodia and in Euglena gracilis and Amoeba proteus cells by urea polyacrylamide gel electrophoresis and activation of cyclic nucleotide phosphodiesterase and of myosin light chain kinase.     

Ras protein of the slime mold Physarum polycephalum is farnesylated in vitro

Physarum Ppras1 protein was efficiently prenylated by prenyltransferases of spinach. Surprisingly in spite of the C-terminal sequence (CLLL) specific for geranylgeranylation the protein was preferentially farnesylated. Consequences of this observation are discussed.     

Some properties of Physarum actinin. A regulatory protein of actin polymerization.

A factor termed Physarum actinin was isolated and partially purified from plasmodia of a myxomycete, Physarum polycephalum. When Physarum actinin was mixed with purified Physarum or rabbit striated muscle G-actin in a weight ratio of about 1 actinin to 9 actin and then the polymerization of G-actin induced, G-actin polymerized to the ordinary F-actin on addition of 0.1 M KCl. However, it polymerized to Mg-polymer on addition of 2 mM MgCl2. The reduced viscosity (etasp/C) of the Mg-polymer was 1.2 dl/g, about one-seventh of that of the F-actin (7.4 dl/g). The sedimentation coefficient of the Mg-polymer was 22.8 S, almost the same as that of the F-actin (29.4 S). The Mg-polymer showed the specific ATPase activity of the order of 1 . 10(-3) mumol ATP/mg actin per min. It was shown that Physarum actinin copolymerized with G-actin to form Mg-polymer on addition of 2 mM MgCl2. The molecular weights of Physarum actinin were about 90 000 in salt-free or slat solutions and 43 000 in a dodecyl sulfate solution. The range of salting out with ammonium sulfate was 50--65% saturation, which was different from that of Physarum actin (15--35% saturation). Physarum actinin did not interact with Physarum myosin or muscle heavy meromyosin. When the weight ratio of actinin to actin increased, the flow birefringence of the formed Mg-polymer decreased, and it became almost zero at the weight ratio of 1 actinin to 5 actin. ATPase activity reached the maximum level (2.2 . 10(-3) mumol ATP/mg actin per min) at the same ratio. On the addition of Physarum actinin to purified Physarum F-actin which had been polymerized on addition of 2 mM MgCl2 the viscosity decreased rapidly, suggesting that the F-actin filaments were broken in the smaller fragments or that they transformed to Mg-polymers. A factor with properties similar to Physarum actinin was isolated from acetone powder of sea urchin eggs.     

Physarum amoebae express a distinct fragmin-like actin-binding protein that controls in vitro phosphorylation of actin by the actin-fragmin kinase.

Amoebae and plasmodia constitute the two vegetative growth phases of the Myxomycete Physarum. In vitro and in vivo phosphorylation of actin in plasmodia is tightly controlled by fragmin P, a plasmodium-specific actin-binding protein that enables actin phosphorylation by the actin-fragmin kinase. We investigated whether amoebal actin is phosphorylated by this kinase, in spite of the lack of fragmin P. Strong actin phosphorylation was detected only following addition of recombinant actin-fragmin kinase to cell-free extracts of amoebae, suggesting that amoebae contain a protein with properties similar to plasmodial fragmin. We purified the complex between actin and this protein to homogeneity. Using an antibody that specifically recognizes phosphorylated actin, we demonstrate that Thr203 in actin can be phosphorylated in this complex. A full-length amoebal fragmin cDNA was cloned and the deduced amino acid sequence shows 65% identity with plasmodial fragmin. However, the fragmins are encoded by different genes. Northern blots using RNA from a developing Physarum strain demonstrate that this fragmin isoform (fragmin A) is not expressed in plasmodia. In situ localization showed that fragmin A is present mainly underneath the plasma membrane. Our results indicate that Physarum amoebae express a fragmin P-like isoform which shares the property of binding actin and converting the latter into a substrate for the actin-fragmin kinase.     

Studies of histidine phosphorylation by a nuclear protein histidine kinase show that histidine-75 in histone H4 is masked in nucleosome core particles and in chromatin

Histone H4 is a good substrate in vitro for the protein histidine kinase activity found both in Physarum polycephalum nuclear extracts and in Saccharomyces cerevisiae cell extracts. However, histone H4 in nucleosome core particles is not a substrate for these kinases. Isolated chromatin was also not a substrate for the protein histidine kinase. The results significantly limit possible interpretations of histidine phosphorylation on histone H4 in vivo and provide a new, sharper focus for future work. In addition, a polynucleotide kinase activity was identified in the Physarum extracts.     

Phosphohistidine is found in basic nuclear proteins of Physarum polycephalum

Nuclear extracts of the true slime mold, Physarum polycephalum, show protein histidine kinase activity towards exogenous histones [(1985) J. Biol. Chem. 260, 16106-16113]. Physarum microplasmodia were labeled with [32P]phosphate in vivo and two basic proteins containing alkali-stable phosphate were detected. The labeled proteins comigrated with Physarum histones H1 (approximately) and H2A and phosphoamino acid analysis showed that each protein contained [32P]-phosphohistidine. The H2A-like protein was also labeled in isolated nuclei incubated with [35S]thio-ATP. We conclude that some Physarum nuclear proteins contain phosphohistidine.     

F-actin bundling protein from Physarum polycephalum: purification and its capacity for co-bundling of actin filaments and microtubules

An F-actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F-actin bundling protein in Physarum extract was passed through a DEAE-cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S-300 HR followed by chromatography on CM-Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS-PAGE, and the molecular weight was estimated to be 52,000. This F-actin bundling protein is referred to as the 52 kDa protein. Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F-actin and microtubule bundles was Ca(2+)-insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross-reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F-actin bundling protein from HeLa cells) (Yamashiro-Matsumura and Matsumura: J. Biol. Chem. 260:5087-5097, 1985). When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co-bundles composed of both filaments formed. This is the first reported example in which an F-actin bundling protein induced co-bundling of actin filaments and microtubules.     

A cell-cycle-dependent protein of Physarum polycephalum revealed by two-dimensional gel electrophoresis.

Silver-stained two-dimensional polyacrylamide gels of Physarum polycephalum extracts have revealed no major changes in protein accumulation during the synchronous nuclear division cycle. However, one protein, or apparent Mr 32 000 and isoelectric point 4.9, shows a reproducible ten-fold increase in total amount between early G2 phase and metaphase. This protein represents about 0.005% of total plasmodial protein.     

Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle of Physarum polycephalum

Monoclonal antibodies raised against the 34-kD nucleolar protein, B-36, from the slime mold Physarum polycephalum have been used to examine the electron microscopic localization of B-36 during the cell cycle in Physarum. During interphase, B-36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B-36, fibrillarin. During mitosis, B-36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA-specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that in Physarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the "NOR" equivalents do not separate during mitosis as in mammalian cells. In addition, the B-36-enriched nucleolar remnants appear to be recycled from one cell cycle to the next.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

类Cyclin A蛋白在多头绒泡菌细胞周期中的定位研究

采用免疫电镜技术对多头绒泡菌(Physarum polycephalum)是否含有类CyclinA蛋白以及该蛋白在有丝分裂周期各时相的定位进行了研究;并以抗CyclinA抗体封闭细胞内源类CyclinA蛋白的方法,探讨类CyclinA蛋白在多头绒泡菌细胞周期中的作用。免疫电镜结果表明,经抗CyclinA抗体标记的实验组细胞中的金颗粒密度明显高于对照组,说明多头绒泡菌细胞中含有类CyclinA蛋白。实验组样品中,细胞核的金颗粒密度很高,而细胞质的金颗粒密度与对照组的相仿,说明多头绒泡菌细胞中的类CyclinA蛋白是核蛋白。细胞核的金颗粒密度在S期最高,G2期的次之,早中期时明显降低,中期和中期以后与对照组的相近。这种金颗粒密度的变化反映了类CyclinA蛋白在细胞周期中的含量变化。以抗CyclinA抗体分别处理S期和G2期的多头绒泡菌细胞,处理后的细胞分别停滞在原来的时相,细胞核形态变得不规则,核内有空洞现象。处于有丝分裂前期的多头绒泡菌细胞经抗CyclinA抗体处理后,细胞核出现畸变。抗体处理结果说明类CyclinA蛋白是参与多头绒泡菌细胞周期多个转换过程调控的种重要蛋白,主要在S期／G2期和G2期／M期的转换以及走出有丝分裂期的进程中发挥作用。     

The nucleolar RNA-binding protein B-36 is highly conserved among plants

The nucleolar protein B-36 is an RNA-associated protein which has a number of properties in common with pre-mRNA-binding proteins (hnRNP proteins). Like the hnRNP proteins, B-36 appears to be evolutionarily conserved among various eukaryotes (protists and several animal species). The conservation of B-36 throughout the plant kingdom has been investigated using a panel of nine monoclonal antibodies previously shown to recognize a minimum of four different epitopes in Physarum B-36, the protein used to generate the monoclonal antibodies. Two of the epitopes (I and III) are widely conserved in 34 kDa proteins (presumably B-36 homologues) from the various species tested (Chlamydomonas, moss, fern, oat, onion, carrot, and bean). Using immunofluorescence localization in moss and carrot protoplasts, the cross-reacting proteins were shown to be restricted to the nucleolus, further confirming their probable homology to B-36. Epitopes I and III are also unique to the B-36 homologues as demonstrated by the failure of any other bands to cross-react. Another epitope (IV) was specifically recognized in the plant B-36 homologues but exhibited greatly reduced affinity for the monoclonal antibody relative to Physarum B-36. The remaining epitope (II), unlike the others, exhibited variable conservation in the plant B-36 homologues and, in addition, was present in several other seemingly unrelated proteins. Finally, several of the plant species exhibited two cross-reacting variants at roughly the 34 kDa position and in at least one of these cases a single monoclonal antibody was able to distinguish between the two variants, a result indicating that the variants do have bona fide structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of muscle and non-muscle actins by casein kinase 1 in vitro

Skeletal muscle and Physarum actins were markedly phosphorylated by casein kinase 1, but not by casein kinase 2. The amount of radioactive phosphate incorporated into muscle actin with 110 units of casein kinase 1 was approx. 0.2 mol per mol of actin, which was significantly greater than those catalyzed using the same number of enzyme units of protein kinase A or protein kinase C. The Km values of casein kinase 1 for muscle and Physarum actins were 0.270 and 0.667 mg/ml, respectively.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

Effects of some protein kinases, cyclic nucleotides and specific inhibitors of phosphorylation on the mitotic cycle of Physarum polycephalum

The true slime mould Physarum polycephalum was treated with various agents by spraying them upon the cell surface 4 hrs before the second synchronous mitosis. The onset of mitosis was considerably approximated after the plasmodium treatment with protein kinases from rat hepatoma or Ph. polycephalum at the late G2 phase. The catalytic and regulatory subunits of cAMP-dependent pig brain protein kinase caused retardation of mitosis, while the holoenzyme, casein kinase and alkaline phosphatase did not affect the timing of mitosis. The cyclic nucleotides and inhibitors of their metabolic enzymes were used to investigate the role of phosphorylation processes in the mitotic cycle.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.