Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

A 14-3-3 protein of Chlamydomonas reinhardtii associated with the endoplasmic reticulum: nucleotide sequence of the cDNA and the corresponding gene and derived amino acid sequence

Two major 14-3-3 proteins of the unicellular green alga Chlamydomonas reinhardtii were purified and partially sequenced. The obtained data show that the 30-kDa isoform predominant in the cytosol is encoded by a previously cloned and sequenced 14-3-3 cDNA whereas the 27-kDa isoform represents a new 14-3-3 protein which is largely associated with the endoplasmic reticulum (ER). Therefore, the corresponding cDNA was cloned and sequenced. The nucleotide sequence of this new cDNA species and the derived amino acid sequence differ considerably from the previously cloned Chlamydomonas 14-3-3 cDNA. The conclusion that the divergent evolution of the corresponding genes must have started rather early as compared to the 14-3-3 genes of other organisms was corroborated by their different genomic organization. The amino acid sequences of both 14-3-3 isoforms were comparatively analysed to find differences which might be responsible for their differential binding to the ER.     

The distribution of membrane-bound 14-3-3 proteins in organelle-enriched fractions of germinating lily pollen

Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.     

Patterns of Starchy Endosperm Acidification and Protease Gene Expression in Wheat Grains following Germination

The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process.     

Cloning and characterization of a cDNA encoding 14-3-3 protein with leaf and stem-specific expression from wheat.

The 14-3-3 proteins, originally described as the mammalian brain proteins, are ubiquitous eukaryotic proteins and have been shown to exert an array of function. A great number of 14-3-3 sequences have been reported in Eudicotyledon. The data of 14-3-3 from the monocotyledon plants, however, are limited. In this report, a 14-3-3 cDNA (designated as Ta14A) was isolated from wheat. An extensive search in GenBank database revealed another 14 14-3-3 isoforms from monocotyledonous plants. These proteins plus 14-3-3 isoforms from Arabidopsis were used for phylogenetic reconstruction, which revealed two groups of 14-3-3 proteins in monocotyledonous plants, namely epsilon and non-epsilon, respectively. The epsilon isoforms were present in monocotyledonous plants. Therefore, the gene duplication to result in an epsilon and non-epsilon isoforms was likely to take place before the speciation of monocotyledon and Eudicotyledon plants. Structural analysis indicated that the different conserved domains and structural characters existed in the monocotyledon 14-3-3 isoforms, which will affect their interaction with other effector proteins. Ta14A was strongly expressed in leaf and stem, undetected in root, suggesting it may have the unique functions within these tissues. These data suggest that structure difference and spatial expression of 14-3-3 will be the important factors to confine its functional specificity.     

The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.     

A plant homologue to mammalian brain 14-3-3 protein and protein kinase C inhibitor.

We have isolated cDNA clones of Spinacea oleracea L. and Oenothera hookeri of 930 and 1017 base pairs, respectively. The open reading frame deduced from the Oenothera sequence codes for a protein of a calculated molecular mass of 29,200. The primary amino acid sequence exhibits a very high degree (88%) of homology to the 14-3-3 protein from bovine brain, and protein kinase C inhibitor from sheep brain. Subsequently the plant protein was partially purified from leaf extract. The partially purified plant protein inhibited protein kinase C from sheep brain in a heterologous assay system. The active fraction consisted of 5-6 different polypeptides of similar molecular size. One of these proteins crossreacted with a peptide-specific antibody against protein kinase C inhibitor protein from sheep brain.     

The 14-3-3 gene expression specificity in response to stress is promoter-dependent

Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms.     

The Cdk-like protein PCTAIRE-1 from mouse brain associates with p11 and 14-3-3 proteins

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk)-like class of proteins, and is localized mainly in the mammalian brain. Using the yeast two-hybrid system we screened a mouse brain cDNA library with PCTAIRE-1 as bait, and isolated several clones coding for the mouse homologs of the following proteins: p11 (also known as calpactin I light chain) and the η, θ (also known asτ) and ζ isoforms of 14-3-3 proteins. We confirmed that these four proteins interact with PCTAIRE-1 by demonstrating the biochemical interactions using the pure recombinant proteins. The fact that 14-3-3 proteins are known to interact with many other intracellular proteins (such as C-kinase, Raf, Bcr, PI3-kinase) and p11 with annexin II (a major pp60^v-src and C-kinase substrate) suggests that PCTAIRE-1 might be part of multiple signal transduction cascades and cellular protein networks. Received: 23 September 1996 / Accepted: 10 January 1997     

Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root

Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.     

Subcellular localization of 14-3-3 proteins in Toxoplasma gondii tachyzoites and evidence for a lipid raft-associated form

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.     

Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco.

Pollen cells are symplasmically isolated during maturation and germination. Pollen therefore needs to take up nutrients via membrane carriers. Physiological measurements on pollen indicate sucrose transport in the pollen tube. A cDNA encoding a pollen-specific sucrose transporter-like protein NtSUT3 was isolated from a tobacco pollen cDNA library. NtSUT3 expression is detected only in pollen and is restricted to late pollen development, pollen germination and pollen tube growth. Altogether these data indicate that pollen is supplied not only with glucose, but also with sucrose through a specific sucrose transporter. The respective contribution of each transport pathway may change during pollen tube growth.     

Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.     

Post-translational modification of 14-3-3 isoforms and regulation of cellular function

14-3-3 is now well established as a family of dimeric proteins that can modulate interaction between proteins involved in a wide range of functions. In many cases, these proteins show a distinct preference for a particular isoform(s) of 14-3-3 and in many cases a specific repertoire of dimer formation influences the particular proteins that 14-3-3 interact. Well over 200 proteins have been shown to interact with 14-3-3. The purpose of this review is to give an overview of the recently identified post-translational modifications of 14-3-3 isoforms and how this regulates function, interaction, specificity of dimerisation between isoforms and cellular location of target proteins. The association between 14-3-3 and its targets usually involves phosphorylation of the interacting protein which has been the subject of many reviews and discussion of this is included in other reviews in this series. However, it is now realised that in some cases the phosphorylation and a number of other, novel covalent modifications of 14-3-3 isoforms may modulate interaction and dimerisation of 14-3-3. Since this aspect is now emerging to be of major importance in the mechanism of regulation by 14-3-3 isoforms and has not been the focus of previous reviews, this will be detailed here.     

棉花143-3L基因的分子鉴定及其在纤维发育中优势表达分析

14-3-3蛋白以二聚体形式存在于所有真核生物中,是一种高度保守的调节蛋白,在细胞生长、增殖、凋亡、信号转导等生命活动中发挥着重要调控作用。我们在棉纤维cDNA文库中分离克隆到1个基因(cDNA),编码14-3-3蛋白类似物,命名为Gh14-3-3L(Gossypiumhirsutum14-3-3-like)。该cDNA长度为1,029bp,包含762bp开放阅读框,其编码蛋白由253个氨基酸组成。Gh14-3-3L与其他真核生物的14-3-3蛋白具有较高的同源性,并具有14-3-3蛋白的基本结构:二聚体结构域、磷酸化丝氨酸富集识别序列、4个CC结构和1个EFHand结构。Northern杂交分析显示Gh14-3-3L在棉纤维发育早期优势表达,且在10DPA棉纤维细胞中表达量最高,这表明Gh14-3-3L基因可能涉及棉纤维细胞伸长过程的调节。研究还表明,该基因在胚珠和花瓣组织中也有较强的表达,但在其他组织中表达较弱或不表达。     

Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers

We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, beta, gamma, epsilon, eta, sigma, tau, and zeta of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of tau isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of beta, epsilon, gamma and eta and a low level of zeta and tau, but not sigma isoform. However, the levels of 14-3-3 epsilon, gamma and eta were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.     

Molecular evolution of the 14-3-3 protein family

Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene fromCaenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2–5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins fromEntamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/ HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed. Correspondence to: D.C. Shakes     

FTICR-MS analysis of 14-3-3 isoform substrate selection

The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client.