Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2.

The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene

The maize locus, Opaque-2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b-32. It is shown that the promoter of the b-32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the 'endosperm box', a motif thought to be involved in endosperm-specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.     

Processing of a chimeric protein in chloroplasts is different in transgenic maize and tobacco plants

Transgenic maize (Zea mays L.) and tobacco (Nicotiana tabacum Petit Havana SR1) plants have been generated, which overproduce a mitochondrial Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD) in chloroplasts. For this, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a Pisum sativum ribulose-1,5-bisphosphate carboxylase (Rubisco) gene and expression of the chimeric gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic MnSOD gene product was correctly targeted to the chloroplasts both in maize and tobacco. However, despite the use of the CaMV 35S promoter, the MnSOD was predominantly localized in the chloroplasts of the bundle sheath cells of maize. Furthermore, the transit peptide was cleaved off at a different position in maize and tobacco.     

Dictyostelium protein kinase C-delta-like protein is localized in the cell nucleus

The molecular mechanism whereby protein kinase C (PKC) molecules transduce signals into the cell nucleus is unknown. In this study, we provide evidence that Dictyostelium discoideum contains PKCδ-like protein that is localized in the nucleus. The Dictyostelium PKCδ-like protein has an apparent molecular mass of 76 kDa. This protein is already highly expressed in vegetative Dictyostelium cells. The expression level remained constant up to 12 h of development, and sharply decreased after 16 h. The PKCδ-like protein is phosphorylated in vivo in response to cAMP and phorbol ester stimulation. Immunofluorescent studies, as well as subcellular fractionation experiments, have indicated that Dictyostelium PKCδ-like protein is permanently located in the nucleus. Our results may indicate that PKCδ-like protein in Dictyostelium functions as a link between cAMP and the tumor-promoting phorbol esters, and events that take place in the nucleus.     

Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant

A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed -zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies.     

The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.

Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing alpha-zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of alpha-zein required simultaneous synthesis of gamma-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presence of gamma-zein, there was a turnover of alpha-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.     

Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.     

Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins.