Novel CK2α and CK2ß subunits in maize reveal functional diversification in subcellular localization and interaction capacity.

In plants, CK2α/ß subunits are encoded by multigenic families. They assemble as heterotetrameric holoenzymes or remain as individual subunits and are usually located in distinct cell compartments. Here we revise the number of maize CK2α/ß genes, bringing them up to a total of eight (four CK2α catalytic and four CK2ß regulatory subunits). We characterize CK2ß4, which presents nuclear localization and interacts with CK2α1, CK2α3, CK2ß1, and CK2ß3. We also describe two CK2α isoforms (CK2α2 and CK2α4) containing N-terminal extensions that correspond to putative cTPs (chloroplast transit peptides). These cTPs are functional and responsible for the subcellular localization of CK2α2 and CK2α4 in chloroplasts. Phylogenetic analysis of the CK2α gene family, further supported by the gene structure and architecture of conserved protein domains, reveals the evolutionary expansion and diversification of this family. The subcellular localization of all four CK2α isoforms was found to be altered when were co-expressed with CK2ß, thereby pointing to the latter as regulators of CK2α localization.

Self-assembly of the amphipathic helix (VHLPPP)8. A mechanism for zein protein body formation.

gamma-Zein, a maize storage protein with an N-terminal proline-rich repetitive domain (gamma-ZNPRD), is located at the periphery of protein bodies. This domain appears to be indispensable for the aggregation of the protein on the surface of the organelle. The peptide (VHLPPP)8, spanning the gamma-ZNPRD, adopts a polyproline II (PPII) conformation that gives an amphipathic helix different from the alpha-helix. We used atomic force microscopy to study the surface organisation of the octamer, and transmission electron microscopy to visualise aggregates of the peptide in aqueous solution. We consider two self-assembly patterns that take account of the observed features. The micellar one fits best with the experimental results presented. Moreover, we found that this peptide has properties associated with surfactants, and form micelles in solution. This spontaneous amphipathic arrangement of the gamma-ZNPRD suggests a mechanism of gamma-zein deposition inside maize protein bodies.

Combined use of ESI-MS and UV diode-array detection for localization of disulfide bonds in proteins: application to an alpha-L-fucosidase of pea.

A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10-30 kDa). The method combines the use of high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) and HPLC with UV diode-array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in alpha-L-fucosidase from pea. The two disulfide bonds were located between residues Cys64 and Cys109 and between Cys162 and Cys169, while Cys127 was free.

Location of disulfide bonds in mature alpha-L-fucosidase from pea.

Fuc-9 is the mature form of a vacuolar alpha-L-fucosidase enzyme which seems to play an important role in plant growth regulation. Fuc-9 is a 202-residue protein containing five Cys residues located at positions 64, 109, 127, 162 and 169. In this study, the disulfide structure of Fuc-9 was determined by MALDI-TOF mass spectrometry (MS), with minimal clean-up of the samples and at a nanomolar scale. Two strategies, based on a specific chemical cleavage (with 2-nitro-5-thiocyanobenzoic acid and alkaline conditions) at the Cys residues and modification of Cys residues by acrylamide/deuterium labeled acrylamide alkylation, were used. Using these methods, the disulfide pairings Cys64-Cys109 and Cys162-Cys169 could be established. The advantages and limitations of our experimental approach are discussed.

Leaf C40.4: a carotenoid-associated protein involved in the modulation of photosynthetic efficiency?

Comparative analysis by differential RNA display (DDRT-PCR) of the expression patterns of potato plants induced and non-induced to tuberize, led to the isolation of a cDNA clone, C40.4, that is strongly upregulated in the leaves of tuberizing plants. Leaf expression of this transcript was shown to be light-dependent, with increased levels of mRNA and protein being detected during the light hours. Sequence analysis revealed near complete identity to potato CDSP34, a thylakoid protein induced by drought stress, and strong homology to the carotenoid-associated proteins fibrillin, ChrB and PAP from pepper fruit, and CHRC from cucumber flowers. By using an antibody against pepper fibrillin, we were able to demonstrate a chloroplastic location of the C40.4 protein in association with the thylakoid membranes. Two-dimensional analysis of thylakoid pigment-protein complexes showed a specific association of the protein with the photosystem II (PSII) multi-subunit complex. Antisense plants with reduced levels of accumulation of C40.4 showed a stunted growth and decreased tuber yield and exhibited reduced values of non-photochemical quenching of chlorophyll a fluorescence. Altogether, these results indicate a preferential association of the C40.4 protein with the light harvesting complex of photosystem II (LHCII) antenna pigment complexes, and suggest a functional role of this protein in photosynthesis, by modulating photosynthetic efficiency and dissipation of excess absorbed light energy within the antenna complex.

Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato. cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide.

A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves. The full 5' region of the cDNA was obtained through a RACE-PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

Lysine-rich gamma-zeins are secreted in transgenic Arabidopsis plants.

We have previously shown that the maize (Zea mays L.) storage prolamine gamma-zein, accumulates in endoplasmic reticulum-derived protein bodies in transgenic plants of Arabidopsis thaliana (L.) ecotype R + P. The retention of gamma-zein in the endoplasmic reticulum was found to be mediated by structural features contained in the polypeptide, an N-terminal proline-rich and a C-terminal cysteine-rich domain which were necessary for the correct retention and assembly of gamma-zein within protein bodies (M.I. Geli et al., 1994, Plant Cell 6: 1911-1922). In the present work we incorporated in the gamma-zein gene lysine-rich coding sequences which were positioned after the N-terminal proline-rich domain and at five amino-acid residues from the C-terminus. The targeting of lysine-rich gamma-zeins was analyzed by expression of chimeric genes regulated by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. The lysine-rich gamma-zeins were detected by immunoblotting and we found that these proteins were modified posttranslationally to reach their mature form. Subcellular fractionation and immunocytochemical studies demonstrated that glycosylated lysine-rich gamma-zeins were secreted to the cell wall of transgenic Arabidopsis leaf cells.

Lysine-rich modified gamma-zeins accumulate in protein bodies of transiently transformed maize endosperms.

During maize seed development, endosperm cells synthesize large amounts of storage proteins, alpha-, beta-, and gamma-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize gamma-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the gamma-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich gamma-zein constructs under the control of the 1.7 kb gamma-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the gamma-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified gamma-zeins accumulate within these organelles and co-localized with endogenous alpha- and gamma-zeins. The studies reported here show the feasibility of manipulating the gamma-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.

Molecular characterization of the gene coding for GPRP, a class of proteins rich in glycine and proline interacting with membranes in Arabidopsis thaliana.

The gene coding for a new class of proteins rich in glycine and proline (GPRP) was cloned in Arabidopsis thaliana. In the protein sequence, five amino acids - glycine, proline, alanine, tyrosine and histidine - account for 79.4% of the total composition. The protein has two different glycine-rich domains interrupted by a hydrophobic segment having a high probability of helix formation. The protein synthesized in vitro interacts with microsomes possibly through the hydrophobic domain. The gene in Arabidopsis has two introns, one in the coding region and the other one in the 5' non-coding region. The later one is 778 bp long. Homologous sequences are found in carrot, tomato and tobacco. GPRP mRNA is found in the different organs of the plant analyzed except in mature seeds and anthers, and mostly in epidermal and vascular tissues. Possible hypotheses about the function of GPRP are discussed.

Two Structural Domains Mediate Two Sequential Events in [gamma]-Zein Targeting: Protein Endoplasmic Reticulum Retention and Protein Body Formation.

[gamma]-Zein is a maize storage protein synthesized by endosperm cells and stored together with [alpha]- and [beta]-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of [gamma]-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for [gamma]-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of [gamma]-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of [gamma]-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of [gamma]-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we observed that only those [gamma]-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of [gamma]-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes.

Role of structural domains for maize gamma-zein retention in Xenopus oocytes.

In order to examine the role of cysteine (Cys)-rich domains in the accumulation of maize (Zea mays L.) gamma-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of gamma-zein and of two truncated forms of gamma-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the gamma-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire gamma-zein and both truncated forms of the gamma-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for gamma-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for gamma-zein targeting on the endoplasmic reticulum.

The Expression Pattern of the Tonoplast Intrinsic Protein gamma-TIP in Arabidopsis thaliana Is Correlated with Cell Enlargement.

The vacuolar membrane (tonoplast) contains an abundant intrinsic protein with six membrane-spanning domains that is encoded by a small gene family. Different isoforms of tonoplast intrinsic protein (TIP) are expressed in different tissues or as a result of specific signals. Using promoter-beta-glucuronidase (GUS) fusions and in situ hybridization, we have examined the expression of gamma-TIP in Arabidopsis thaliana. GUS staining of plants transformed with promoter-GUS fusions showed that gamma-TIP gene expression is high in recently formed tissues of young roots. In the shoot, gamma-TIP gene expression was highest in the vascular bundles of stems and petioles, as well as in the stipules and in the receptacle of the flower. No GUS activity was detected in root or shoot meristems or in older tissues, suggesting temporal control of gamma-TIP gene expression associated with cell elongation and/or differentiation. In situ hybridization carried out with whole seedlings confirmed that in root tips, gamma-TIP mRNA was present only in the zone of cell elongation just behind the apical meristem. In seedling shoots, mRNA abundance was also found to be correlated with cell expansion. These results indicate that gamma-TIP may be expressed primarily at the time when the large central vacuoles are being formed during cell enlargement.

Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana.

Reports from a number of laboratories describe the presence of a family of proteins (the major intrinsic protein family) in a variety of organisms. These proteins are postulated to form channels that function in metabolite transport. In plants, this family is represented by the product of NOD26, a nodulation gene in soybean that encodes a protein of the peribacteroid membrane, and tonoplast intrinsic protein (TIP), an abundant protein in the tonoplast of protein storage vacuoles of bean seeds (KD Johnson, H Höfte, MJ Chrispeels [1990] Plant Cell 2: 525-532). Other homologs that are induced by water stress in pea and in Arabidopsis thaliana and that are expressed in the roots of tobacco have been reported, but the location of the proteins they encode is not known. We now report the presence and derived amino acid sequences of two different TIP proteins in A. thaliana. alpha-TIP is a seed-specific protein that has 68% amino acid sequence identity with bean seed TIP; gamma-TIP is expressed in the entire vegetative body of A. thaliana and has 58% amino acid identity with bean seed TIP. Both proteins are associated with the tonoplast. Comparisons of the derived amino acid sequences of the seven known plant proteins in the major intrinsic protein family show that genes with similar expression patterns (e.g. water stress-induced or seed specific) are more closely related to each other than the three A. thaliana homologs are related. We propose that the nonoverlapping gene expression patterns reported here, and the evolutionary relationships indicated by the phylogenetic tree, suggest a functional specialization of these proteins.

Purification, characterization and immunological properties of 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize (Zea mays) seeds.

2,3-Bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) was purified and characterized from maize. SDS electrophoresis showed only one band with a molecular mass of 64 kDa, similar to that determined for the native enzyme by gel-filtration chromatography. The kinetic constants were similar to those reported for wheat germ phosphoglycerate mutase. Rabbit antiserum against maize phosphoglycerate mutase possesses a high degree of specificity. It also reacts with the wheat germ enzyme but fails to react with other cofactor-independent or cofactor-dependent phosphoglycerate mutases. Cell-free synthesis experiments indicate that phosphoglycerate mutase from maize is not post-translationally modified.

Immunological properties of rat phosphoglycerate mutase isozymes.

In mammalian tissues three phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) isozymes result from the homo-dimeric and hetero-dimeric combinations of two subunits (types M and B). Whereas rabbit antisera against type M subunit (purified from rat muscle) and against type BB isozyme (purified from rat brain) possessed a high degree of specificity, both antisera reacted with type BB and MM isozymes, as demonstrated by immunoneutralization and ELISA. Both the M subunit and B subunit were more immunoreactive than their respective dimeric isozymes. Subunits type M and B may possess common antigenic determinants, and some of these determinants may be sterically hindered in their dimeric structures.

Signal recognition-like particles are present in maize.

We show that maize storage protein translocation across microsomal membranes is mediated by signal recognition particles (SRPs) similar to those described in animal systems (Dobberstein, B. (1978) Hoppe-Seyler's Z. Physiol. Chem. 252, 955-962; Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116). We have prepared a high salt extract from endosperm cell homogenates, from which a ribosome-free fraction was obtained. This fraction is enriched in an SRP-like factor which apparently corresponds to a ribonucleoprotein particle that sediments at about 12 S. The RNA moiety of this 12 S particle is complex, showing a three-band electrophoretic pattern and sedimenting at about 8 S. The fraction restores translocation competence of salt-washed maize microsomes as tested by using a pre-zein message. In contrast to canine SRPs, the maize SRP-like component does not cause a translation arrest of maize storage proteins (zein) in a wheat germ cell-free system.