Green algal microtubule-associated protein with a molecular weight of 90 kDa which bundles microtubules

Summary Cytoplasmic streaming in the freshwater, coencytic green alga,Dichotomosiphon tuberosus, is regulated by light. Conspicuous changes are observed in the number of microtubules cross-linked together in bundles when the cytoplasmic streaming is modulated by light. In an attempt to identify the cross-linker, we stainedD. tuberosus cells with antibodies specific for several different microtubules-associated proteins (MAPs) from vertebrates. Antibodies raised against bovine adrenal 190 kDa MAP stained the algal cells, and the pattern of staining was quite similar to that obtained with tubulin-specific antibodies. Examination by immunoelectron microscopy revealed that the antibodies specific for the 190 kDa microtubule-associated protein (MAP) were located along the microtubules. Western blotting demonstrated that the antibodies crossreacted with a peptide fromD. tuberosus with a molecular weight of about 90 kDa. This peptide was heat-stable, a property shared by the bovine 190 kDa MAP. Moreover, this 90 kDa peptide, crossreacted with antibodies raised against a synthetic peptide, identical to the tubulin-binding domain found in the 190 kDa MAP and in a tau protein. Partially purified 90 kDa protein fromD. tuberosus has the ability to bundle microtubules when mixed with a tubulin fraction fromD. tuberosus, in the presence of taxol. These results suggest that the 90 kDa protein fromD. tuberosus is a MAP that bundles microtubules.Abbreviations APMSF (p-amidinophenyl) methanesulfonyl fluoride - BSA bovine serum albumin - CBB Coomassie Brilliant Blue R - DEAE diethylaminoethyl - DMSO dimethyl sulfoxide - DOC deoxycholic acid - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MAP microtubule-associated protein - MES 2[N-morpholino] ethanesulfonic acid - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - TLCK N-p-tosyl-lysine chloromethyl ketone     

Modulation by the ratio S-adenosylmethionineS-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. $\text{S-}\text{Adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio $\text{S-}\text{adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ can modulate phosphorylation of a specific protein.     

The structure and function of the 33 kDa extrinsic protein of Photosystem II: A critical assessment

In this review the structure and function of the 33 kDa protein of Photosystem II is examined. Significant controversies exist concerning the solution secondary structure of the protein, the location of its binding site(s) within Photosystem II, the amino acid residues of the 33 kDa protein required for binding and its stoichiometry within the photosystem. The studies which examine these topics are considered from a critical perspective. A hypothetical model of the folding of the 33 kDa extrinsic protein which is supported by site-specific labeling studies and site-directed mutagenesis experiments is presented. Additionally, the function of the protein within the photosystem is unclear. We present a hypothesis that the 33 kDa protein is involved in maintaining the chloride associated with photosynthetic oxygen evolution in close proximity to the oxygen-evolving site.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Modulation by the ratio of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio can modulate phosphorylation of a specific protein.     

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes

Microtubule-associated proteins (MAPs) interact with tubulinin vitro andin vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of -tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells. Microinjection studies along with studies on the cell distribution suggest that MBD-205 appears to play a structural role at the level of the microtubule interactions in these cells.     

Characterisation of the PsbX protein from Photosystem II and light regulation of its gene expression in higher plants

The location and expression of the previously uncharacterised photosystem II subunit PsbX have been analysed in higher plants. We show that this protein is a component of photosystem II (PSII) core particles but absent from light-harvesting complexes or PSII reaction centres. PsbX is, however, localised to the near vicinity of the reaction centre because it can be cross-linked to cytochrome b559, which is known to be associated with the D1/D2 dimer. We also show that the expression of this protein is tightly regulated by light, since neither protein nor mRNA is found in dark-grown plants.     

Increased sulfur amino acids in soybean plants overexpressing the maize 15 kDa zein protein

Summary Four transgenic soybean [Glycine max (L.) Merrill] lines were generated containing the maize 15 kDa zein protein gene using somatic embryogenic protocols. The zein gene was inserted behind the β-phaseolin promoter for seed-specific expression. All four lines represent separate transformation events as they were generated in different experiments at different locations. Two of the transformation events produced multiple plants, and these produced identical Southern hybridization patterns (UKY/Z1, UKY/Z2 and UKY/Z3 from the first; and OSU/Z4, OSU/Z8 and OSU/Z10 from the second). Molecular characterization revealed that multiple copies of the zein gene were present in all of the transgenic lines. Immunoblot analysis confirmed the accumulation of the zein protein product in the seeds of the UKY/Z1, UKY/Z2, UKY/Z3, OSU/Z4, OSU/Z8 and OSU/Z10 transgenic lines. However, there was no accumulation of zein protein in the UGA/Z1 line and Northern analysis confirmed that the zein transgene was silenced in this line. It was not possible to analyze the zein expression in the seeds of the UKY/Z4 line, as it was sterile. Amino acid analysis of the UKY and OSU lines confirmed that there was a 12–20% increase in methionine, and 15–35% increase in cysteine content in these lines compared to the control. There were no consistent changes in the content of the other amino acids in the transgenic lines. These results suggest that while the increase in methionine content in these lines is modest, it is possible to increase the methionine content without adversely affecting the protein composition of soybean.     

Distribution,pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine

Background information. The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high‐affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. Results. TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam‐binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5‐4864 and protoporphyrin IX showed 5–13‐fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage‐dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium‐dependent chloride secretion in the duodenum and calcium‐dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Conclusions. The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.     

A role for shoot protein in shoot-root dry matter allocation in higher plants

BACKGROUND AND AIMS: It is stated in many recent publications that nitrate (NO3-) acts as a signal to regulate dry matter partitioning between the shoot and root of higher plants. Here we challenge this hypothesis and present evidence for the viewpoint that NO3- and other environmental effects on the shoot:root dry weight ratio (S:R) of higher plants are often related mechanistically to changes in shoot protein concentration. METHODS: The literature on environmental effects on S:R is reviewed, focusing on relationships between S:R, growth and leaf NO3- and protein concentrations. A series of experiments carried out to test the proposal that S:R is dependent on shoot protein concentration is highlighted and new data are presented for tobacco (Nicotiana tabacum). KEY RESULTS/EVIDENCE: Results from the literature and new data for tobacco show that S:R and leaf NO3- concentration are not significantly correlated over a range of environmental conditions. A mechanism involving the relative availability of C and N substrates for growth in shoots can explain how shoot protein concentration can influence shoot growth and hence root growth and S:R. Generally, results in the literature are compatible with the hypothesis that macronutrients, water, irradiance and CO2 affect S:R through changes in shoot protein concentration. In detailed studies on several species, including tobacco, a linear regression model incorporating leaf soluble protein concentration and plant dry weight could explain the greater proportion of the variation in S:R within and between treatments over a wide range of conditions. CONCLUSIONS: It is concluded that if NO3- can influence the S:R of higher plants, it does so only over a narrow range of conditions. Evidence is strong that environmental effects on S:R are often related mechanistically to their effects on shoot protein concentration.     

Fluorescence and Fourier-transform infrared spectroscopic studies on the role of disulfide bond in the calcium binding in the 33 kDa protein of Photosystem II

The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca²⁺ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD circular dichroism - FTIR Fourier transform infrared - La lanthanum - PS photosystem - Tb terbium     

A 67-kDa plasma-membrane-bound Ca2+-stimulated protein kinase active in sink tissue of higher plants

A novel 67-kDa protein kinase (p67 ^cdpk ) was identified in the microsomal membrane fraction of apple (Malus domestica Borkh. cv. Braeburn) suspension cultures and subsequently found to be active in sink tissues. Microsomal proteins were blotted onto Nylon or polyvinylidenedifluoride membranes, and p67 ^cdpk assayed by in situ-labelling renatured proteins with [γ-³²P]ATP; thin-layer electrophoresis/thin-layer chromatography of acid hydrolysates of the ³²P-labelled protein band indicated that serine and threonine, but not tyrosine residues were phosphorylated. A detailed analysis of the ion-dependency of p67 ^cdpk revealed that it was a Ca²⁺-stimulated, Mg²⁺-dependent protein kinase. However, p67 ^cdpk was ten times more active in the presence of 10 mM Mn²⁺, and these assay conditions were used routinely to increase the sensitivity of the assay. Activity of p67 ^cdpk was found at high levels in the plasma membrane, and solubilisation experiments with a number of detergents suggested that p67 ^cdpk is an integral membrane protein. A homologous protein kinase with similar biochemical properties was also present in cell-suspension cultures of pear and maize. In maize (Zea mays L.) plants, sink tissues, such as young expanding leaves of both light-grown and etiolated plants, mature etiolated tissue and roots all had high levels of p67 ^cdpk activity. However, mature light-grown (source) tissues had barely detectable levels. In etiolated maize leaves and coleoptiles the kinase activity was highest in expanding tissue and decreased as the cells expanded. When etiolated maize plants were exposed to light, the activity of p67 ^cdpk was reduced to background levels after 8 h. Although p67 ^cdpk has biochemical properties similar to those of other plant calcium-dependent protein kinases, this is the first identification of a membrane-bound calcium-dependent protein kinase which is specifically active in sink tissues. Received: 14 July 1997 / Accepted: 25 September 1997     

Isolation, characterization and expression of a cDNA coding for a jasmonate-inducible protein of 37 kDa in barley leaves

In barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate-inducible proteins (JIPs). In the present study, a new jasmonate-inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate-treated barley leaf segments. The open reading frame (ORF) encodes a 39-9 kDa polypeptide which cross-reacts with antibodies raised against the in vivo JIP-37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C-terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP-37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP-23. The expression pattern of the JIP-37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α-linolenic acid and 12-oxophytodienoic acid, we hypothesize that there is a stress-induced lipid-based signalling pathway in which an endogenous rise of jasmonate switches on JIP-37 gene expression. Using immunocytochemical techniques, JIP-37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.     

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle.     

Cd2+、Al3+作用下蚕豆UDS与微核相关性分析及高等植物UDS技术初探

为探讨金属离子对高等植物非按期DNA合成(UnscheduledDNASythesis,简称UDS)和微核(MCN)的诱导作用、二者之间的关联性以及利用高等植物UDS技术检测环境诱变物的可行性,利用3HTdR前体掺入法研究了Cd²⁺、Al³⁺作用下蚕豆的UDS效应。结果表明,Cd²⁺、Al³⁺均能不同程度地诱导蚕豆UDS和MCN的发生;UDS量与微核率(MCNF)之间呈负相关(r<0),但相关不显着(|r|0.05),且二者间的相关程度在Cd²⁺和Al³⁺两种金属离子作用下没有显着差别(P>0.05);利用高等植物UDS技术检测环境诱变物质,在一定受检物剂量范围内是可靠的,但超过这个剂量范围,UDS技术无法检出.     

Heat sensitivity of Rubisco,Rubisco activase and Rubisco binding protein in higher plants

During the past few years the investigations concerning Rubisco and the changes of its activity and properties at elevated temperature were reconsidered with special reference to the important role of Rubisco activase and Rubisco binding protein. The major changes in Rubisco, Rubisco activase and Rubisco binding protein reported recently are presented in this review. New information on these proteins, including their changes under heat stress conditions, is discussed together with open questions.     

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129 ) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST–ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.     

A radial system of microtubules extending between the nuclear envelope and the plasma membrane during early male haplophase in flowering plants

The angiosperm male meiocyte is unusual among plant cells in that during early development ordered cellulosic microfibrils are not deposited at the protoplast surface. Instead, a complex series of events takes place which leads to the formation of the pollen wall primexine. The use of immuno-cytochemistry, electron microscopy and experiments with an inhibitor have revealed this cell to contain no cortical microtubules, and its cytoskeleton to be radially organised with microtubules extending from the nucleus to the plasma membrane. It is proposed that these microtubules, perhaps organised from the nuclear envelope, play a part in orientation of the nucleus and in the transport of materials to the cell surface.