PROTEIN RETENTION IN THE ENDOPLASMIC RETICULUM OF INSECT CELLS IS NOT COMPROMISED BY BACULOVIRUS INFECTION

High level expression of the major auxin-binding protein (ABP1) from maize (Zea maysL.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immuno-localization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.     

Molecular analysis of auxin-specific signal transduction

The auxin-binding protein (ABP1) of maize has been purified, cloned and sequenced. Homologues have been found in a wide range of plants and at least seven ABP sequences from four different species are now known. We have developed a range of anti-ABP antibodies and these have been applied to analysis of the structure, localization and receptor function of ABP. ABP1 is a glycoprotein with two identical subunits of apparent M _r =22 kDa. The regions recognised by our five monoclonal antibodies (MAC 256–260) and by polyclonal antisera from our own and other laboratories have been specified by epitope mapping and fragmentation studies. All polyclonal anti-ABP sera recognise two or three dominant epitopes around the single glycosylation site. Two monoclonals (MAC 256, 259) are directed at the endoplasmic reticulum (ER) retention sequence KDEL at the C-terminus. Early biochemical data pointed to six amino acids likely to be involved in the auxin binding site. Inspection of the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containing five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, suggesting that the region is highly conserved. This is confirmed by more recent information showing that the selected polypeptide contains the longest stretch of wholly conserved sequence in ABP1. Most strikingly, the antibodies show auxin agonist activity against protoplasts in three different electrophysiological systems-hyperpolarization of tobacco transmembrane potential; stimulation of outward ATP-dependent H⁺ current in maize; modulation of anion channels in tobacco. The biological activity of these antibodies indicates that the selected peptide does form a functionally important part of the auxin binding site and strongly supports a role for ABP1 as an auxin receptor. Although ABP contains a KDEL sequence and is located mainly in the ER lumen, the electrophysiological evidence shows clearly that some ABP must reach the outer face of the plasma membrane. One possible mechanism is suggested by our earlier demonstration that the ABP C-terminus recognised by MAC 256 undergoes an auxin-induced conformational change, masking the KDEL epitope and it is of interest that this C-terminal region appears to be important in auxin signalling [22]. So far we have been unable to detect the secretion of ABP into the medium of maize cell (bms) cultures reported by Jones and Herman [7]. However, recent silver enhanced immunogold studies on maize protoplasts have succeeded in visualizing ABP at the cell surface, as well as auxin-specific clustering of the signal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membrane and gene levels remain to be elucidated.     

Authentic processing and targeting of active maize auxin-binding protein in the baculovirus expression system.

The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.     

Retention of maize auxin-binding protein in the endoplasmic reticulum: quantifying escape and the role of auxin

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested one proposed model to account for release of ABP1 from the ER, namely that auxin binding induces a conformational change in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretion of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected under all conditions. Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants. Received: 31 October 1996 / Accepted: 16 December 1996     

Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein

The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots.     

KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall

The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane.     

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3–5 pmol/mg protein with an apparent dissociation constant (K _d) between 3 and 5 μM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22–24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31–33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-³H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity

There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 μg ABP1 per gram of fresh leaf. This value is comparable to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants. The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene. Received: 24 February 1999 / Accepted: 25 March 1999     

In vitro binding affinities of 4-chloro-, 2-methyl-, 4-methyl-, and 4-ethylindoleacetic acid to auxin-binding protein 1 (ABP1) correlate with their growth-stimulating activities

4-Chlorindole-3-acetic acid (4-CI-IAA), an endogenous auxin in certain plant species of Fabaceae, has a higher efficiency in stimulating cell elongation of grass coleoptiles compared with indole-3-acetic acid (IAA), particularly at low concentrations. However, some investigations reported a 1,000-fold discrepancy between growth stimulation and binding affinity of 4-CI-IAA to auxin-binding protein 1 (ABP1) from maize. Here we report binding data of 4-CI-IAA and three alkylated IAA derivatives using purified ABP1 in equilibrium dialysis. There is a clear correlation between the growth-promoting effects and the binding affinity to ABP1 of the different IAA analogues measured by competition of [³H]naphthalene-1-acetic acid binding. Our data are consistent with the hypothesis that ABP1 mediates auxin-induced cell elongation.Abbreviations ABP1 auxin-binding protein 1 - 4-CI-IAA 4-chloroindole-3-acetic acid - NAA naphthalene-1-acetic acid - ER endoplasmic reticulum - IAA indole-3-acetic acid - 2-Me-IAA 2-methylindole-3-acetic acid - 4-Me-IAA 4-methylindole-3-acetic acid - 4-Et-IAA 4-ethylindole-3-acetic acid - MES 4-morpholineethanesulfonic acid - PAA phenylacetic acid     

Cycling of auxin-binding protein through the plant cell: pathways in auxin signal transduction.

The auxin receptor literature contains a glaring discrepancy that invites explanation. While some physiological experiments suggest that active auxin receptors are sited inside the cell, others point to action at the cell surface. Furthermore, although the major auxin-binding protein (ABP) of maize (Zea mays) coleoptiles is found in the lumen of the endoplasmic reticulum (ER), exogenous ABP can mediate auxin-dependent changes in the plasma membrane potential of protoplasts. How can an ER protein mediate changes in cell potential? To resolve this dilemma, I propose that ABP cycles through the cell. In response to auxin, ABP is released from the ER and follows a secretory pathway to the cell surface. After secretion, ABP would bind sites on the cell surface and become subject to endocytosis, cycling back to the ER. Elevated auxin would accelerate the cycling of ABP between the ER and the cell surface. If cell wall precursors interacted with ABP during their progression through the secretory pathway, this would provide a mechanism for regulating cell wall synthesis. At the cell surface ABP would regulate an enzyme responsible for maintaining membrane potential. Both of these responses are components of auxin-regulated growth. This hypothesis does not exclude other mechanisms of signal transduction, particularly in gene regulation.     

Diacidic motif is required for efficient transport of the K+ channel KAT1 to the plasma membrane

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K(+) channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.     

Auxin perception and signal transduction

The action of auxin on whole plants is very complex, but we are starting to understand how some of the earliest events are signalled in single cells. There is now good evidence that auxin induces rapid events at the plasma membrane by binding to a population of the auxin-binding protein ABPI, which is associated with a membrane-spanning docking protein, possibly a G-protein-coupled receptor (GPCR). ABPI is targeted to the endoplasmic reticulum (ER) lumen, but it does not appear to bind auxin within the ER and its function (if any) in this location is unknown. It is also not known how the protein reaches the cell surface, but it is possible that it is exported together with its docking protein. Binding of auxin causes a conformational change affecting the C-terminus of ABPI and it is likely that this change serves to activate the receptor at the plasma membrane. The signal transduction pathway appears to involve activation of phospholipase A₂(PLA₂) leading to the production of lipid second messengers which activate the plasma membrane proton ATPase (H⁻-ATPase) by a phosphorylation-dependent mechanism. Branch points exist that could potentially lead from this pathway to responses in the nucleus, but there is not yet any firm evidence that ABP1 is involved in such responses. Since intracellular auxin concentrations are correlated with sensitivity in some cases, it is possible that there is also a site of auxin perception inside the cell.     

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.     

生长素结合蛋白cDNA的克隆及其在烟草中的表达

基于拟南芥内质网生长素结合蛋白基因的cDNA序列,设计合成了Ap5和Ap3两个引物,应用RT－PCR技术扩增了拟南芥的ABP基因。将该基因克隆在植物表达载体p35SSIN的35S启动子和Nos3’端之间,得到植物表达载体p35SE。通过农杆菌个导的方法对烟草SR1进行了转化,由分子杂交等检测证明,生长素结合蛋白基因已在烟草中表达,同时转基因烟草后代对生长素的敏感性明显增加。     

The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum.

The yeast alpha1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man9GlcNAc2 to a single isomer of Man8GlcNAc2. alpha1,2-Mannosidase is a 63 kDa type II resident membrane protein of the endoplasmic reticulum that has none of the known endoplasmic reticulum localization signals (HDEL/KDEL, KKXX, or RRXX). Using antibodies against recombinant alpha1,2-mannosidase, indirect immunofluorescence showed that alpha1,2-mannosidase localization is abnormal in rer1 cells and that the alpha1,2-mannosidase localizes in the vacuoles of rer1/deltapep4 cells whereas in wild-type and deltapep4 cells it is found in the endoplasmic reticulum. 35S-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha1,2-mannosidase, then with either alpha1,2-mannosidase antibodies or antibodies to alpha1,6-mannose residues added in the Golgi. The labeled proteins were examined by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A significant proportion of the labeled alpha1,2-mannosidase was immunoprecipitated by alpha1,6-mannose antibodies in wild-type, deltapep4 and rer1/deltapep4 cells with endogenous levels of alpha1,2-mannosidase, and in wild-type, deltapep4, rer1 and rer1/deltapep4 cells overexpressing alpha1,2-mannosidase. The alpha1,2-mannosidase of rer1/deltapep4 cells had a slower mobility on the gels than alpha1,2-mannosidase precipitated from wild-type or deltapep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles. It is concluded that the endoplasmic reticulum localization of alpha1,2-mannosidase in wild-type cells depends on Rer1p for retrieval from an early Golgi compartment.     

Plasmalemma hyperpolarity and culture of sense and antisense abp transgenic tobacco protoplasts

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Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.     

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c

The auxin-binding protein 1 (ABP1) has already been proved to be an extracellular receptor of auxin in single cell systems. Protoplasts of maize coleoptiles respond to auxin with an increase in volume. The 2-naphthaleneacetic acid (2-NAA), an inactive auxin analog, acts as an anti-auxin in protoplast swelling, as it suppresses the effect of indole-3-acetic acid (IAA). Antibodies raised against box a of ABP1 induce protoplast swelling in the absence of auxin. This response is inhibited by pre-incubation with 2-NAA. The effect of 2-NAA on swelling induced by agonistic antibodies appears to depend on the binding characteristics of the antibody. ScFv12, an antibody directed against box a, box c and the C-terminal domain of ABP1 also exhibits auxin-agonist activity which is, however, not abolished by 2-NAA. Neither does 2-NAA affect the activity of the C-terminal peptide of ABP1, which is predicted to interact with putative binding proteins of ABP1. These results support the view that box a and box c of ABP1 are auxin-binding domains.