Margarita Torrent

Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17 164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279–2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.

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.

Eukaryotic protein production in designed storage organelles

BackgroundProtein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.ResultsVarious Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain.ConclusionThe Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.

Subcellular localization of glutelin-2 in maize (Zea mays L.) endosperm.

Accumulation of the 28 KD protein of the glutelin-(G2) fraction was followed in developing maize endosperm, using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peak integration of scanned gels. 28 KD glutelin-2 could already be observed from 15 days after pollination and its accumulates reached a plateau during the second half of the development period. The process of biosynthesis of 28 KD glutelin-2 and zeins occurs in a parallel way. Subcellular fractions obtained from linear sucrose gradient centrifugation of developing maize endosperms were analyzed by SDS-PAGE and immunoblotting using a serum reacting against glutelin-2 and 14 KD Z2. Glutelin-2 was found to be present in the protein bodies when subcellular fractionation was carried out without dithiothreitol (DTT). The presence of a reducing agent causes the elution of glutelin-2 from protein bodies. Immunocytochemical labelling using the protein A-colloidal gold technique in protein bodies incubated with anti-G2 IgG revealed that G2 is located mainly in the periphery of protein bodies. These results are interpreted as indicating a structural role for glutelins in protein bodies.

Expression of genes for cell-wall proteins in dividing and wounded tissues of Zea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.

Protein Body Induction: A New Tool to Produce and Recover Recombinant Proteins in Plants

Stable accumulation of storage proteins, lipids and carbohydrates is a hallmark of the plant seed, and is a characteristic that is typically deficient in existing platforms for recombinant protein manufacture. One of the biological sequestration mechanisms that facilitate the folding, assembly and stabilization of plant seed storage proteins involve the de novo formation of unique intracellular organelles, the endoplasmic reticulum (ER)-derived protein bodies (PBs). In cereals, such as maize, PBs are formed directly in the lumen of the ER of endosperm cells and contain zeins, a group of polypeptides, which account for more than half of the total seed protein mass. The 27 kD gamma zein protein localizes to the periphery of the PBs surrounding aggregates of other zeins (including a zein and delta zein). Heterologous expression of gamma zein has been shown to result in the formation of PB-like structures, and the N-terminal proline-rich domain of gamma zein (Zera), containing eight PPPVHL repeats and a Pro-X sequence is by itself capable of directing ER retention and PB formation in non-seed tissues. We present a novel approach to produce recombinant proteins in plants based on the ability of gamma zein-Zera domain to store recombinant proteins inside PBs. Zera domain fused to several proteins, including a enhanced cyan fluorescent protein (ECFP), calcitonin (Ct) and epidermal growth factor (EGF), were cloned into vectors for transient or stable transformation of tobacco plants. In tobacco leaves, we observed the formation of dense, ER-localized structures containing high concentrations of the respective target proteins. The intact synthetic organelles containing Zera fusions were readily isolated from cellular material using density-based separation methods.

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, α-, β, and γ-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 γ-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the γ-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich γ-zein constructs under the control of the 1.7 kb γ-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 γ-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 γ-zeins accumulate within these organelles and co-localized with endogenous - and γ-zeins. The studies reported here show the feasibility of manipulating the γ-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.

The maize Dof protein PBF activates transcription of γ-zein during maize seed development

Maize PBF (prolamin-box binding factor) belongs to the Dof class of plant specific transcription factors containing one highly conserved zinc finger DNA-binding domain, called Dof (DNA binding with one finger) domain. Maize PBF trans-activates the γ-zein gene (γZ) promoter in developing maize seeds as shown by transient expression in maize endosperms. Co-transfection of a γZ:GUS construct with 35S:PBF resulted in a sevenfold increase in GUS expression, however, PBF mutation in Cys residues within the Dof domain abolishes both, binding to DNA and the capacity to activate γZ promoter. We present two pieces of evidence that PBF transactivates γZ promoter by binding to the Pb3 motif (TGTAAAG). First, recombinant Dof domain of PBF (bdPBF) specifically recognized Pb3 site as shown by gel mobility shift assays and second, co-expression of PBF with γZ promoter mutated in Pb3 motif suppressed PBF trans-activation capacity. Immunocytochemical analysis on developing endosperm sections shows that PBF is localized in the nuclei of the peripheral layer cells of starchy endosperm, the tissue in which the initial accumulation of γ-zein protein occurs. By contrast, PBF is detected in the cytosol of the starchy endosperm cells newly differentiated from aleurone daughter cells, where γ-zein was absent. Taken together these data indicate that maize PBF plays an essential role in the regulation of the temporal and spatial expression of γZ gene.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)8 of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4–6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys7 and Cys9) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

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

In order to examine the role of cysteine (Cys)-rich domains in the accumulation of maize (Zea mays L.) γ-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of γ-zein and of two truncated forms of γ-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the γ-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 γ-zein and both truncated forms of the γ-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 γ-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for γ-zein targeting on the endoplasmic reticulum.