BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.     

Characterization of cis-regulatory regions responsible for developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana

The Arabidopsis GA1 gene encodes copalyl diphosphate synthase, which catalyzes the first committed step in the gibberellin biosynthetic pathway. Previous studies indicated that the expression pattern of the GA1 gene is tissue-specific and cell-type-specific during development. Here we showed that expression of GA1 cDNA driven by the 2.4 kb 5-upstream sequence plus the GA1 genomic coding region into the third exon was able to rescue the ga1-3 mutant phenotype. To understand the mechanism controlling GA1 gene expression, cis-regulatory regions in the GA1 promoter were identified by promoter deletion analysis with the GA1--glucuronidase (GUS) gene fusion system. The second intron and the region from –1391 to –997, with respect to the translation initiation site, positively regulate overall GA1-GUS expression level in all tissues examined. Several additional regulatory regions are involved in GA1-GUS expression in all the stages except in seeds: two positive regulatory regions in the first intron and the sequence between –425 and –207, and a negative regulatory region between –1848 and –1391. We also found that the region between –997 and –796 is essential for a high level of GA1 expression in developing seeds.     

Distinct repressing modules on the distal region of the <Emphasis Type="Italic">SBP2</Emphasis> promoter contribute to its vascular tissue-specific expression in different vegetative organs

The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position −2000 to −700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (−1785/−1508), Frag III (−1507/−1237), Frag IV (−1236/−971) and Frag V (−970/−700), act independently to alter the constitutive pattern of −92pSBP2-mediated GUS expression in different organs. Frag V fused to −92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions −1485 to −1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive −92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter.     

The promoter of an antifungal protein gene from Gastrodia elata confers tissue -specific and fungus-inducible expression patterns and responds to both salicylic acid and jasmonic acid

Gastrodia antifungal proteins (GAFPs) are a group of mannose-binding lectins purified from Gastrodia elata that show strong resistance against a wide spectrum of fungi. The GAFP-2 promoter was analyzed for its ability to control the expression of the reporter gene, -glucuronidase (GUS) in transgenic tobacco plants. The GUS assays revealed that the GAFP-2 promoter is expressed in a tissue-specific manner, which mainly expressed in the vascular cells. The highest GUS activity was observed in roots, followed by stems. GAFP-2-GUS expression was strongly induced by the fungus Trichoderma viride and by the plant stress regulators, salicylic acid and jasmonic acid in the stably transformed tobacco plants. The –537 region of the GAFP-2 promoter was sufficient for its tissue-specific and inducible expression of the promoter.Communicated by H.S. Judelson     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between –330 to –200, was shown to be necessary for nitrate induction of -glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5 deletions in the–330 to –200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to –230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between –230 to –200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the –300 to –130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the –230 to –181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The –240 to –110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.     

The Endoplasmic Reticulum Chaperone BiP/GRP78 Is Important in the Structure and Function of the Human Cytomegalovirus Assembly Compartment

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP''s KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP''s KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.Human cytomegalovirus (HCMV), the largest of the human herpesviruses, is capable of encoding over 200 proteins, which are expressed in temporal fashion as immediate-early, early, delayed-early, and late genes. Despite the extensive coding capacity of HCMV, its replication cycle is slow. During this protracted period, the virus must maintain optimal replication conditions in the host cell. However, the increasing strain of the infection induces cellular stress responses with consequences that may be deleterious to the progress of the infection. We and others have previously shown that HCMV has multiple mechanisms to deal with the deleterious aspects of cellular stress responses while maintaining beneficial ones (2, 8-10, 14, 17, 18, 22-24, 26, 27, 50, 51).An example of these mechanisms is the viral control of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Due to the number of HCMV proteins that are glycosylated, or receive other ER-dependent posttranslational modifications, the load of proteins in the ER can exceed its capacity, resulting in ER stress and the activation of the UPR (18, 47, 51). However, we and others have shown that HCMV controls and modulates the UPR, maintaining aspects that may benefit the viral infection while inhibiting aspects that would be detrimental (18, 51).The UPR is normally controlled by transmembrane sensors which initiate the complex UPR signaling cascade when activated by ER stress (reviewed in references 20, 35, 38, and 52). The ER molecular chaperone BiP (immunoglobulin heavy chain-binding protein), also called glucose-regulated protein 78 (GRP78), is believed to bind these sensors and keep them inactive during unstressed conditions. However, when unfolded or misfolded proteins accumulate in the ER, BiP leaves these sensors to perform its chaperone function, thus allowing the sensors to activate UPR signaling. We have previously shown that during HCMV infection, BiP is vastly overproduced (8), suggesting that BiP may have other functions in the viral infection. Indeed, it has been shown that BiP binds to the viral proteins US2 and US11; this interaction is necessary for the virus-mediated degradation of major histocompatibility complex class I and II (15, 47). Further, we have shown that depletion of BiP, using either the BiP-specific subtilase cytotoxin SubAB (32) or short hairpin RNAs, caused infectious virion formation in the cytoplasm to cease and nucleocapsids to accumulate just outside the outer nuclear membrane (8). This result suggested that BiP has a significant role in virion formation and cytoplasmic egress.Although the exact mechanism of virion formation in the cytoplasm is not well understood, studies have identified a perinuclear structure, referred to as the cytoplasmic assembly compartment, that is involved in the process. Several viral proteins, for example, tegument proteins (pp28, pp65) (36) and viral glycoproteins (gB, gH, gL, gO, gp65) (36, 46), have been identified as part of this structure. Defining the exact origin of this compartment has been complicated by the observation of specific organellar markers in and around the compartment, while other markers of the same organelles are not detected. For example, immunofluorescence examination suggests that the early endosomal marker early endosome antigen 1 (EEA1) has been observed in the center of the assembly compartment (12, 13); however, Rab4 and Rab5, other early endosomal markers, were not detected (16). Such observations suggest that the virus directs specific viral and cellular proteins to the assembly compartment as needed for assembly compartment function.In the present study, we further examine the role of BiP during an HCMV infection, including its localization and interactions with other proteins. We show here that in infected cells, BiP localizes in two distinct structures, regions of condensed ER near the periphery of the cell and the assembly compartment. The data suggest that BiP diversion from the ER to the assembly compartment is due to occlusion of its ER localization signal. Depletion of BiP causes both condensed ER and assembly compartments to disperse, indicating that BiP is important for their formation or maintenance. BiP and pp28 appear to associate in the assembly compartment, since BiP from the assembly compartment coimmunoprecipitates pp28 and vice versa. In addition, both BiP and pp28 copurify with the assembly compartment on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous studies (1, 4) have shown that cells infected with HCMV with a mutation in the TRS1 gene show cytoplasmic and assembly compartment defects like those seen when BiP is depleted (reference 8 and the studies presented below). We show that a fraction of TRS1 purifies with the assembly compartment, indicating a shared assembly compartment function with BiP. In summary, our data suggest that BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.     

Molecular Chaperone BiP Interacts with Borna Disease Virus Glycoprotein at the Cell Surface

Borna disease virus (BDV) is characterized by highly neurotropic infection. BDV enters its target cells using virus surface glycoprotein (G), but the cellular molecules mediating this process remain to be elucidated. We demonstrate here that the N-terminal product of G, GP1, interacts with the 78-kDa chaperone protein BiP. BiP was found at the surface of BDV-permissive cells, and anti-BiP antibody reduced BDV infection as well as GP1 binding to the cell surface. We also reveal that BiP localizes at the synapse of neurons. These results indicate that BiP may participate in the cell surface association of BDV.Borna disease virus (BDV) belongs to the Bornaviridae family of nonsegmented, negative-strand RNA viruses and is characterized by highly neurotropic and noncytopathic infection (18, 33). BDV infects a wide variety of host species and causes central nervous system (CNS) diseases in animals, which are frequently associated with behavioral disorders (14, 19, 29, 31). BDV cell entry is mediated by endocytosis, following the attachment of viral envelope glycoprotein (G) to the cellular receptor (2, 7, 8). BDV G is translated as a precursor protein, GP, which is posttranslationally cleaved by the cellular protease furin to generate two functional subunits of the N (GP1) and C (GP2) termini (28). Recent studies revealed that GP1 is involved in virus interaction with as-yet-unidentified cell surface receptor(s) and that GP2 mediates a pH-dependent fusion event between viral and cell membranes (2, 7, 27). In addition, a previous work using a hippocampal culture system suggested that BDV G is required for viral dissemination in neurons (2); however, cellular factors involved in BDV cell entry, especially cell surface association, remain to be elucidated.To extend our understanding of the role of BDV G in the interaction with the cell plasma membrane, we transfected GP1 fused with hemagglutinin-tobacco etch virus protease cleavage site-FLAG tags (GP1-TAP) into human oligodendroglioma OL cells. GP1-TAP was purified using anti-FLAG M2 affinity gel (Sigma). To verify that GP1-TAP binds to OL cells, the cells were incubated with 4 μg/ml GP1-TAP, and binding was detected by anti-FLAG M2 antibody (Sigma). A flow cytometric analysis indicated that GP1-TAP binds to OL cells (Fig. ​(Fig.1A).1A). To further validate the binding of GP1-TAP, we tested whether GP1-TAP inhibits BDV infection. OL cells were pretreated with 4 μg/ml GP1-TAP for 30 min. Proteins purified from mock-transfected cells using an anti-FLAG M2 affinity gel served as a control. The cells were then mixed with cell-free BDV. After 1 h of absorption, the supernatants were removed and fresh medium was added. At 3 days postinfection, the viral antigens were stained with anti-nucleoprotein (N) monoclonal and anti-matrix (M) polyclonal antibodies. As shown in Fig. ​Fig.1B,1B, GP1-TAP reduced BDV infection by 40% compared to levels for mock-treated cells. This result was consistent with earlier reports showing that recombinant GP1 protein binds to the cell surface and inhibits BDV infection (6, 20).Open in a separate windowFIG. 1.BDV GP1 binds to the cell surface. (A) Binding of BDV GP1 to OL cells. OL cells were incubated with GP1-TAP (solid line), and its binding was detected using anti-FLAG M2 antibody and flow cytometry. As a control, cells incubated with proteins purified from mock-transfected cells were detected by an anti-FLAG M2 antibody (dotted line). (B) Inhibition of BDV infection by GP1. OL cells pretreated with GP1-TAP were inoculated with the BDV huP2br strain. Values are the means + standard deviations (SD) from three independent experiments. **, P < 0.01.To investigate the host factor(s) that mediates the interaction of GP1 with the cell surface, a combination of tandem affinity purification (TAP) and liquid chromatography tandem mass spectrometry analyses was designed (13). We transfected GP1-TAP into OL cells and then purified GP1 from cell homogenates using a TAP strategy. We compared the purified proteins from the whole-cell and cytosol fractions (Fig. ​(Fig.2A),2A), and the bands detected only in the whole-cell fraction were determined as GP1-binding proteins in the membrane and/or nuclear fractions. In addition to GP1 protein (Fig. ​(Fig.2A,2A, arrow), we identified a specific band around 80 kDa in the whole-cell homogenate, but not in the cytosol fraction (Fig. ​(Fig.2A,2A, arrowhead), and determined that the band corresponded to the BiP (immunoglobulin heavy chain-binding protein) molecular chaperone, also called glucose-regulated protein 78 (GRP78), by mass spectrometry analysis. We confirmed the specific interaction between endogenous BiP and BDV G in infected cells by immunoprecipitation analysis (Fig. ​(Fig.2B).2B). To map the binding domain on BiP to GP1, we constructed a series of deletion mutants of the green fluorescent protein (GFP)-tagged BiP plasmid (Fig. ​(Fig.2C).2C). We transfected the mutant plasmids into BDV-infected OL cells and then performed an immunoprecipitation assay using anti-GFP antibody (Invitrogen). As shown in Fig. ​Fig.2D,2D, BDV G was coimmunoprecipitated with truncated BiP mutants, except for BiPΔN-GFP, which lacks the ATP-binding domain of BiP (lane 3), suggesting that BiP interacts with GP1 via its N-terminal region.Open in a separate windowFIG. 2.BDV GP1 interacts with BiP molecular chaperone. (A) TAP analysis of BDV GP1. Proteins coimmunoprecipitated with GP1-TAP in OL cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Cyt, cytosol fraction; Wc, whole-cell homogenate. Arrow, GP1-TAP; arrowhead, BiP. (B) Coimmunoprecipitation (IP) of BDV G and endogenous BiP. BDV G was immunoprecipitated from BDV-infected OL cells by anti-BDV G polyclonal antibody. Endogenous BiP was then detected by anti-BiP monoclonal antibody (Becton Dickinson). IgG, immunoglobulin G. (C) Schematic representation of deletion mutants of recombinant BiP-GFP. The known functional regions are indicated. (D) Immunoprecipitation analysis of BiP-GFP mutants in BDV-infected OL cells. The deletion plasmids were transfected and immunoprecipitated by anti-GFP antibody. Specific binding was detected using anti-BDV G antibody. Lane 1, GFP; lane 2, BiP-GFP; lane 3, BiPΔN-GFP; lane 4, BiPΔPB-GFP; lane 5, BiPΔC-GFP.BiP is known to be resident primarily in the endoplasmic reticulum and functions as a molecular chaperone involved in the folding process of nascent proteins, mostly through interaction with its peptide-binding domain (12, 17, 21). On the other hand, BiP has been reported to serve as a coreceptor of certain viruses at the plasma membrane (15, 34). Recent studies also revealed that cell surface BiP mediates the internalization of its ligands into cells (1, 10). We first investigated whether BiP is expressed on the cell surface of BDV-permissive OL and 293T cells using an anti-BiP polyclonal antibody (H-129; Santa Cruz Biotechnology, Inc.). As shown in Fig. ​Fig.3A,3A, BiP expression is detected on the surface of both cell lines. This result is in agreement with recent observations that BiP is expressed on the surface of various types of cells (9, 10, 15, 23, 24, 34). We also investigated whether BiP is expressed on the cell surface of BDV-nonpermissive cell lines, such as HeLa and CHO cells. As shown in Fig. ​Fig.3A,3A, we detected BiP expression on the surface of HeLa, but not CHO, cells. These observations were confirmed by immunofluorescence analysis (Fig. ​(Fig.3B).3B). Note that BiP is clearly detected at the endoplasmic reticulum in the permeabilized CHO cells by the antibody (see Fig. S1 in the supplemental material), suggesting that BiP is expressed at a very low level, if at all, on the surface of CHO cells. We next examined whether cell surface BiP serves as a binding molecule of BDV GP1. To test this, we performed an inhibition assay using an anti-BiP polyclonal antibody (N-20; Santa Cruz Biotechnology, Inc.) which recognizes the N terminus of BiP. As shown in Fig. ​Fig.3C,3C, the antibody inhibited GP1 binding to the cell surface by 40%. Furthermore, BDV infection was found to decrease by 70% when cells were treated with the antibody (Fig. ​(Fig.3D3D).Open in a separate windowFIG. 3.Cell surface BiP mediates cell association of BDV. (A) Flow cytometric analysis was performed with anti-BiP antibody (H-129) in BDV-permissive (OL and 293T) and -nonpermissive (HeLa and CHO) cells (solid lines). Cells stained with normal rabbit immunoglobulin G were used as a control (dotted lines). (B) Immunofluorescence analysis was performed by using anti-BiP antibody (H-129) with BDV-permissive and -nonpermissive cells. Arrows indicate BiP staining at the membrane. Scale bars, 10 μm. (C) Inhibition of GP1 binding by anti-BiP antibody (N-20). OL cells were pretreated with anti-BiP antibody, followed by labeling with GP1. GP1 binding on the cell surface was detected using flow cytometry. Values are the means + SD from three independent experiments. *, P < 0.05. (D) Inhibition of BDV infection by anti-BiP antibody. OL cells were incubated with 10 μg/ml anti-BiP antibody or normal goat immunoglobulin G and then the cells were mixed with cell-free BDV. After 1 h absorption, the supernatants were replaced with fresh medium. Virus infection was measured by immunofluorescence analysis using anti-N and -M antibodies at 3 days postinfection. Values are the means + SD from three independent experiments. *, P < 0.05. IgG, immunoglobulin G.To investigate the role of cell surface BiP in the infection of BDV, the BiP expression was inhibited by short interfering RNA (siRNA) in OL cells (see Fig. S2A in the supplemental material). We selected an siRNA (Hs_HSPA5_4; Qiagen, Inc.) which could partially downregulate the cell surface expression of BiP (see Fig. S2B in the supplemental material). However, siRNA treatment of BiP did not influence the infectivity of BDV in OL cells (see Fig. S2C in the supplemental material). This may be due to an incomplete reduction of BiP expression on the cell surface. Alternatively, while BiP mediates at least in part the cell surface association of BDV particles, this result may exhibit the presence of another, as-yet-unidentified BDV G-binding protein that is involved in the binding and subsequent cell entry of BDV.Previous studies demonstrated that BDV can be traced centripetally and transsynaptically after olfactory, ophthalmic, or intraperitoneal inoculation (3, 25). Migration of BDV to the CNS after footpad infection can be prevented by sciatic nerve transection (3). These observations suggest that BDV may disseminate primarily via neural networks. Recently, it has been demonstrated that BDV G was expressed at the termini of neurites or at contact sites of neurites (2), suggesting that local assembly of BDV may take place at the presynaptic terminals of synapses, similar to assembly of other neurotropic viruses (22, 26, 32). If BiP localizes at synapse sites, BiP may efficiently participate in the transmission of BDV particles at the synapses. To evaluate this hypothesis, we examined BiP localization in primary culture of mouse hippocampal neurons. After in vitro culture for 17 days, BiP localization was determined by an immunofluorescence assay without permeabilization. As shown in Fig. ​Fig.4A,4A, BiP signals were clearly detected at neurites, including the contact sites between dendrites and axons, as punctate staining (arrows), suggesting that BiP is expressed at the neuronal surface, most likely at the synapses. We next examined the localization of BiP with postsynaptic density 95 (PSD-95), a marker of postsynaptic density (5). Although BiP signals were detected mainly in the perinuclear area of the hippocampal neurons, punctate staining was also found at neurites colocalized with PSD-95 (Fig. ​(Fig.4B,4B, arrows). Taken together, these observations suggested that BiP is distributed at the synaptic surface, including the postsynaptic membrane, of neurons, a possible site for BDV budding and entry (2).Open in a separate windowFIG. 4.BiP localizes at the synaptic surface of hippocampus neurons. (A) Localization of BiP at synaptic surface. Hippocampal neurons were immunostained with anti-BiP antibody (N-20) without permeabilization. A differential interference contrast (DIC) image is shown. Dotted lines in the Merge panel indicate the dendrite outline. Arrows indicate BiP staining at the contact sites between axons and dendrites. (B) Colocalization between BiP and a postsynaptic protein. Hippocampal neurons were immunostained with anti-BiP (N-20) and anti-PSD-95 (Millipore) antibodies. Arrows indicate colocalized signals of BiP and PSD-95 at neurites. Scale bars, 10 μm.In summary, this study demonstrates that BiP is a GP1-binding protein at the synaptic surface. This is the first report showing the BDV G-binding factor on the cell surface. The first step of BDV entry might be mediated by the interaction of GP1 with as-yet-unidentified cell surface receptors, which may form a complex with other molecules, such as BiP. We showed that treatment with anti-BiP antibody affects BDV infection as well as GP1 binding to the cell surface (Fig. ​(Fig.3).3). Furthermore, synaptic distribution of BiP was found in hippocampal primary neurons (Fig. ​(Fig.4).4). These findings strongly suggest that BiP plays critical roles in BDV association with the neuronal surface via interaction with GP1. On the other hand, a BDV-nonpermissive cell line, HeLa, appeared to express BiP on the cell surface, suggesting that the cell surface BiP may not be necessarily involved in the infectivity of BDV. A recent study by Clemente et al. (6) revealed that following initial attachment to the cell surface, BDV is recruited to the plasma membrane lipid raft (LR) prior to internalization of the particles. The study suggested that BDV may use the LR as a platform to interact with additional host cell factor(s) required for efficient BDV internalization. Because BiP does not contain transmembrane regions, BiP needs another host protein(s) with transmembrane regions on the cell surface. It has been reported that cell surface BiP interacts with diverse proteins, such as major histocompatibility complex class I molecules (34), the voltage-dependent anion channel (9), and the DnaJ-like protein MTJ-1 (4), all of which associate with LR in the plasma membrane (16, 24, 35). Once BDV has attached to the cell surface, it might utilize such BiP-associated LR proteins for efficient cell surface attachment or internalization. Previously, it has been proposed that kainate 1 (KA-1) receptor might represent the BDV receptor within the CNS (11). Because some glutamate receptors are shown to bind to BiP (30), KA-1 receptors might interact with BiP and serve as a receptor complex for BDV. Further studies are required for a full understanding of the cell association processes, especially receptor binding, of BDV.      

Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum

We have previously shown by RNA gel blot analyses that the tobacco polyubiquitin-encoding gene Ubi.U4 is expressed in a complex pattern during plant development (Genschik et al., 1994). In order to study its tissue-specific expression, we cloned the fragment containing the –263 bp proximal promoter of the gene, the leader intron and the first ubiquitin monomer in front of the reporter GUS gene. Histochemical analyses for GUS activity during tobacco plant development revealed that the gene is expressed at variable amounts in many plant tissues with high levels in metabolically active and/or dividing cells and in the vascular tissues of the plant. We also analysed the expression pattern of constructs in which either the intron or the intron together with the first ubiquitin monomer were deleted. Our results indicate that the ubiquitin leader intron is not only a quantitative determinant of gene expression but may also influence the tissue-specific expression pattern.     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

Ntlim1, a PAL-box binding factor,controls promoter activity of the horseradish wound-inducible peroxidase gene

To understand molecular mechanisms underlying wound-induced expression of plant peroxidase genes, the promoter of a horseradish C2 peroxidase (prxC2) gene was analyzed. We had previously isolated a tobacco nuclear protein, Ntlim1, as a trans factor binding to a PAL-box motif of the prxC2 promoter; however, the function of the Ntlim1 trans factor and the PAL-box motif in wound-responsive expression of the prxC2 gene remains unclear. Here, we found that the prxC2 promoter without the intact PAL-box motif failed to direct a normal level of both the basal and the wound-induced expression of -glucuronidase (GUS) reporter gene in transgenic tobacco plants, indicating that the PAL-box motif functions as an essential cis element of the prxC2 promoter. We also found that antisense expression of Ntlim1 in transgenic plants carrying the prxC2 promoter::GUS chimeric construct decreased not only the level of the basal and the wound-induced expression of the GUSreporter gene but also the extent of wound inducibility of the prxC2 promoter itself. This result indicates that Ntlim1 is required for the basal level of prxC2 promoter activity as well as its up-regulation under wound stress. Moreover, consistent with the results obtained in planta, result from super-shift assay indicates that the Ntlim1 binds to the PAL-box motif independently of wound stress.     

The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Detection of the putative<Emphasis Type="Italic"> cis-</Emphasis>region involved in the induction by a<Emphasis Type="Italic"> Pyricularia oryzae</Emphasis> elicitor of the promoter of a gene encoding phenylalanine ammonia-lyase in rice

A rice PAL (phenylalanine ammonia-lyase) gene sequence (rPAL-P5), which is highly similar to and likely the same as a previously described rice ZB8PAL gene, including the 5-upstream and exon I coding regions of PAL, was isolated using PCR amplification. The expression of several PALs, including rPAL-P5, was strongly induced following inoculation with Pyricularia oryzae or treatment with a P. oryzae elicitor. To identify the promoter region induced by the P. oryzae elicitor, we constructed and subsequently transformed rPAL-P5 promoter deletion series into rice calli using particle bombardment. Results from both elicitor-inducible reporter gene and gel mobility shift assays demonstrated that the sequence –349 to –256 of the rPAL-P5 promoter includes a cis-element involved in the induction of P. oryzae.Abbreviations CTAB Cetyltrimethylammonium bromide - 2,4-D 2,4-Dichlorophenoxyacetic acid - GUS -Glucuronidase - 4-MU 4-Methylumbelliferone - 4-MUG 4-Methylumbelliferyl glucuronide - NOS Nopaline synthase - PAL Phenylalanine ammonia-lyase Communicated by J.C. Register III     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5 ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5 region of the MVK gene to the -glucuronidase (GUS) reporter gene, indicated that the MVK 5-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 5-flanking region are crucial for high-level MVK gene expression.