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ER bodies are endoplasmic reticulum (ER)-derived organelles specific to the order Brassicales and are thought to function in plant defense against insects and pathogens. ER bodies are generally classified into two types: constitutive ER bodies in the epidermal cells of seedlings, and wound-inducible ER bodies in rosette leaves. Herein, we reveal a third type of ER body found in Arabidopsis ( Arabidopsis thaliana ) rosette leaves and designate them "leaf ERbodies" (L-ER bodies). L-ER bodies constitutively occurred in specific cells of the rosette leaves: marginal cells, epidermal cells covering the midrib, and giant pavement cells. The distribution of L-ER bodies was closely associated with the expression profile of the basic helix-loop-helix transcription factor NAI1, which is responsible for constitutive ER-body formation. L-ER bodies were seldom observed in nai1 mutant leaves, indicating that NAI1 is involved in L-ER body formation. Confocal imaging analysis revealed that L-ER bodies accumulated two types of β-glucosidases: PYK10, the constitutive ER-body β-glucosidase; and BETA-GLUCOSIDASE18 (BGLU18), the wound-inducible ER-body β-glucosidase. Combined with the absence of L-ER bodies in the bglu18 pyk10 mutant, these results indicate that BGLU18 and PYK10 are the major components of L-ER bodies. A subsequent feeding assay with the terrestrial isopod Armadillidium vulgare revealed that bglu18 pyk10 leaves were severely damaged as a result of herbivory. In addition, the bglu18 pyk10 mutant was defective in the hydrolysis of 4-methoxyindol-3-ylmethyl glucosinolate These results suggest that L-ER bodies are involved in the production of defensive compound(s) from 4-methoxyindol-3-ylmethyl glucosinolate that protect Arabidopsis leaves against herbivory attack., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Endoplasmic reticulum (ER) bodies are thought to function in plant defense against insects and pathogens. Recently, a new type of ER body referred to as "leaf ER bodies" (L-ER bodies) was identified in Arabidopsis rosette leaves. L-ER bodies accumulate two β-glucosidases, namely PYK10 and BGLU18, which are characteristic of previously described constitutive ER bodies and inducible ER bodies, respectively. However, it is unclear how the biogenesis of L-ER bodies, which are similar to both constitutive and inducible ER bodies, is regulated. In the present study, we show that the biogenesis of L-ER bodies is regulated by both jasmonate (JA)-dependent and -independent pathways. Confocal imaging analysis revealed the presence of L-ER bodies in the JA insensitive mutant coronatine insensitive 1-1 ( coi1-1 ), which lacks the JA receptor COI1. Quantitative reverse transcription polymerase chain reaction analysis revealed that the expression of BGLU18 mainly depends on the JA signaling pathway while that of PYK10 does not. In addition, expression of the ER body related genes NAI1, NAI2 , and TSA1 was reduced in the coi1-1 mutant relative to the wild type. Taken together, these findings suggest that JA signaling is not necessary for the formation of L-ER bodies, while it is partially required for gene expression of L-ER body components.

The endoplasmic reticulum (ER) is a large network made of membranous cisternae and tubules, which accounts for a large proportion of the total lipid bilayer endomembrane of the cell. In mammals and yeast, LUNAPARK proteins are preferentially localized at the three-way junctions of the ER network, stabilizing the junctions and establishing the ER architecture. We identified two Arabidopsis homologs and designated them LNPA and LNPB. Subcellular localization analysis with a non-dimerizable type of green fluorescent protein (GFP) revealed that both LNPA and LNPB are predominantly distributed throughout the ER, but not preferentially localized at the three-way junctions. Quantitative analysis of the network in the double mutant lnpa lnpb revealed that deficiency of LNPA and LNPB caused the cortical ER to develop poor ER cisternae and a less dense tubular network. These phenotypes are opposite to those of LNP-deficient mutants of yeast and mammals. Despite the importance of cysteine residues in the zinc finger motif of the yeast LNP homolog (Lnp1p), the corresponding cysteine residues of LNPA were not necessary for the stabilization of ER morphology because replacing the four cysteine residues in the zinc finger motif of the LNPA protein with alanine residues did not affect its function. A significant phenotype of lnpa lnpb is generation of large spherical structures from the ER. Formation of the structures might reduce the amounts of the ER membrane to be used for generating the network, resulting in poor development of the ER network. Taken together, our results suggest that plant LNPs function differently from those in yeast and mammals: they function to distribute ER membranes appropriately throughout the cells.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

In Arabidopsis thaliana, MAIGO 2 (MAG2) is involved in protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus via its association with the ER-localized t-SNARE components SYP81/AtUfe1 and SEC20. To characterize the molecular machinery of MAG2-mediated protein transport, we explored MAG2-interacting proteins using transgenic A. thaliana plants expressing TAP-tagged MAG2. We identified three proteins, which were designated as MAG2-INTERACTING PROTEIN 1-3 [MIP1 (At2g32900), MIP2 (At5g24350) and MIP3 (At2g42700)]. Both MIP1 and MAG2 localized to the ER membrane. All of the mag2, mip1, mip2 and mip3 mutants exhibited a defect in storage protein maturation, and developed abnormal storage protein body (MAG body) structures in the ER of seed cells. These observations suggest that MIPs are closely associated with MAG2 and function in protein transport between the ER and Golgi apparatus. MIP1 and MIP2 contain a Zeste-White 10 (ZW10) domain and a Sec39 domain, respectively, but have low sequence identities (21% and 23%) with respective human orthologs. These results suggest that the plant MAG2-MIP1-MIP2 complex is a counterpart of the triple-subunit tethering complexes in yeast (Tip20p-Dsl1p-Sec39p) and humans (RINT1-ZW10-NAG). Surprisingly, the plant complex also contained a fourth member (MIP3) with a Sec1 domain. There have been no previous reports showing that a Sec1-containing protein is a subunit of ER-localized tethering complexes. Our results suggest that MAG2 and the three MIP proteins form a unique complex on the ER that is responsible for efficient transport of seed storage proteins., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A-green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to approximately 3.5 microm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.

The endoplasmic reticulum (ER) is composed of tubules, sheets, and three-way junctions, resulting in a highly conserved polygonal network in all eukaryotes. The molecular mechanisms responsible for the organization of these structures are obscure. To identify novel factors responsible for ER morphology, we employed a forward genetic approach using a transgenic Arabidopsis thaliana plant (GFP-h) with fluorescently labeled ER. We isolated two mutants with defects in ER morphology and designated them endoplasmic reticulum morphology1 (ermo1) and ermo2. The cells of both mutants developed a number of ER-derived spherical bodies, approximately 1 microm in diameter, in addition to the typical polygonal network of ER. The spherical bodies were distributed throughout the ermo1 cells, while they formed a large aggregate in ermo2 cells. We identified the responsible gene for ermo1 to be GNOM-LIKE1 (GNL1) and the gene for ermo2 to be SEC24a. Homologs of both GNL1 and SEC24a are involved in membrane trafficking between the ER and Golgi in yeast and animal cells. Our findings, however, suggest that GNL1/ERMO1 and SEC24a/ERMO2 have a novel function in ER morphology in higher plants.

Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated an Arabidopsis thaliana mutant, maigo2 (mag2), that accumulated the precursors of two major storage proteins, 2S albumin and 12S globulin, in dry seeds. mag2 seed cells contained many novel structures, with an electron-dense core that was composed of the precursor forms of 2S albumin. 12S globulins were segregated from 2S albumin and were localized in the matrix region of the structures together with the ER chaperones lumenal binding protein and protein disulfide isomerase, which were more abundant in mag2 seeds. The MAG2 gene was identified as At3g47700, and the MAG2 protein had a RINT-1/TIP20 domain in the C-terminal region. We found that some MAG2 molecules were peripherally associated with the ER membrane. MAG2 had an ability to bind to two ER-localized t-SNAREs (for target-soluble NSF [N-ethylmaleimide-sensitive fusion protein] attachment protein receptor; At Sec20 and At Ufe1). Our findings suggest that MAG2 functions in the transport of storage protein precursors between the ER and Golgi complex in plants.

Plant cells develop various endoplasmic reticulum (ER)-derived structures with specific functions. The ER body, a novel ER-derived compartment in Arabidopsis, is a spindle-shaped structure (approximately 10 microm long and approximately 1 microm wide) that is surrounded by ribosomes. Similar structures were found in many Brassicaceae plants in the 1960s and 1970s, but their main components and biological functions have remained unknown. ER bodies can be visualized in transgenic Arabidopsis expressing the green fluorescent protein with an ER-retention signal. A large number of ER bodies are observed in cotyledons, hypocotyls and roots of seedlings, but very few are observed in rosette leaves. Recently nai1, a mutant that does not develop ER bodies in whole seedlings, was isolated. Analysis of the nai1 mutant reveals that a beta-glucosidase, called PYK10, is the main component of ER bodies. The putative biological function of PYK10 and the inducibility of ER bodies in rosette leaves by wound stress suggest that the ER body functions in the defense against herbivores.

The endoplasmic reticulum (ER) body is a characteristic structure derived from ER and is referred to as a proteinase-sorting system that assists the plant cell under various stress conditions. Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing green fluorescent protein fused with an ER retention signal. ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. We found that wound stress induced the formation of many ER bodies in rosette leaves. ER bodies were also induced by treatment with methyl jasmonate (MeJA), a plant hormone involved in the defense against wounding and chewing by insects. The induction of ER bodies was suppressed by ethylene. An electron microscopic analysis showed that typical ER bodies were induced in the non-transgenic rosette leaves treated with MeJA. An experiment using coi1 and etr1-4 mutant plants showed that the induction of ER bodies was strictly coupled with the signal transduction of MeJA and ethylene. These results suggested that the formation of ER bodies is a novel and unique type of endomembrane system in the response of plant cells to environmental stresses. It is possible that the biological function of ER bodies is related to defense systems in higher plants.

Flavonoids are a major group of plant-specific metabolites that determine flower and seed coloration. In plant cells, flavonoids are synthesized at the cytosolic surface of the endoplasmic reticulum and are sequestered in the vacuole. It is possible that membrane trafficking, including vesicle trafficking and organelle dynamics, contributes to flavonoid transport and accumulation. However, the underlying mechanism has yet to be fully elucidated. Here we show that the Arabidopsis ECHIDNA protein plays a role in flavonoid accumulation in the vacuole and protein trafficking to the vacuole. We found defective pigmentation patterns in echidna seed, possibly caused by reduced levels of proanthocyanidins, which determine seed coloration. The echidna mutant has defects in protein sorting to the protein storage vacuole as well as vacuole morphology. These findings indicate that ECHIDNA is involved in the vacuolar trafficking pathway as well as the previously described secretory pathway. In addition, we found a genetic interaction between echidna and green fluorescent seed 9 (gfs9), a membrane trafficking factor involved in flavonoid accumulation. Our findings suggest that vacuolar trafficking and/or vacuolar development, both of which are collectively regulated by ECHIDNA and GFS9, are required for flavonoid accumulation, resulting in seed coat pigmentation. [ABSTRACT FROM AUTHOR]

Summary: Synaptotagmin 1 (SYT1) has been recognised as a tethering factor of plant endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (EPCSs) and partially localises to around plasmodesmata (PD). However, other components of EPCSs associated with SYT1 and functional links between the EPCSs and PD have not been identified.We explored interactors of SYT1 by immunoprecipitation and mass analysis. The dynamics, morphology and spatial arrangement of the ER in Arabidopsis mutants lacking the EPCS components were investigated using confocal microscopy and electron microscopy. PD permeability of EPCS mutants was assessed using a virus movement protein and free green fluorescent protein (GFP) as indicators.We identified two additional components of the EPCSs, SYT5 and SYT7, that interact with SYT1. The mutants of the three SYTs were defective in the anchoring of the ER to the PM. The ER near the PD entrance appeared to be weakly squeezed in the triple mutant compared with the wild‐type. The triple mutant suppressed cell‐to‐cell movement of the virus movement protein, but not GFP diffusion.We revealed major additional components of EPCS associated with SYT1 and suggested that the EPCSs arranged around the PD squeeze the ER to regulate active transport via PD. [ABSTRACT FROM AUTHOR]

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) in a precursor form and then are transported to protein storage vacuoles (PSVs) where they are converted to their mature form. To understand the mechanisms by which storage proteins are transported, we screened Arabidopsis maigo mutants to identify those that abnormally accumulate storage protein precursors. Here we describe a new maigo mutant, maigo 4 (mag4), that abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag4 seed cells abnormally develop a large number of novel structures that exhibit a highly electron-dense core. Some of these structures were surrounded by ribosomes. Immunogold analysis suggests that the electron-dense core is an aggregate of 2S albumin precursors and that 12S globulins are localized around the core. The MAG4 gene was identified as At3g27530, and the MAG4 protein has domains homologous to those found in bovine vesicular transport factor p115. MAG4 molecules were concentrated at cis-Golgi stacks. Our findings suggest that MAG4 functions in the transport of storage protein precursors from the ER to the Golgi complex in plants. In addition, the mag4 mutant exhibits a dwarf phenotype, suggesting that MAG4 is involved in both the transport of storage proteins and in plant growth and development. [ABSTRACT FROM PUBLISHER]

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) as larger precursors and are sorted to protein storage vacuoles, where they are converted into the mature forms. We report here an Arabidopsis mutant, maigo 1 (mag1), which abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag1 seeds mis-sort storage proteins by secreting them from cells. mag1 seeds have smaller protein storage vacuoles in the seeds than do wild-type seeds. The MAG1 gene encodes a homolog of the yeast (Saccharomyces cerevisiae) protein VPS29. VPS29 is a component of a retromer complex for recycling a vacuolar sorting receptor VPS10 from the pre-vacuolar compartment to the Golgi complex. Our findings suggest that MAG1/AtVPS29 protein is involved in recycling a plant receptor for the efficient sorting of seed storage proteins. The mag1 mutant exhibits a dwarf phenotype. A plant retromer complex plays a significant role in plant growth and development. [ABSTRACT FROM AUTHOR]

Soluble endoplasmic reticulum (ER)-resident proteins have very long lives because of their ER residency. This residency depends largely on ER-retrieval signals at their C-terminus. We examined the long-term destiny of endogenous ER-resident proteins, a lumenal binding protein (BiP) and a protein disulfide isomerase (PDI), with cultured cells of Arabidopsis. ER residents, in contrast to vacuolar proteinases, were considerably degraded in cells at the stationary phase. A subcellular fractionation analysis suggested that ER residents were transported into the vacuoles, which accumulated the residents lacking the ER-retrieval signals. We showed that the PDI located in the vacuoles had high mannose glycans, but not complex glycans, which suggested that the ER resident was transported to the vacuoles independent of the medial/ trans-Golgi complex. To visualize the pathway of transport of ER-resident proteins, tobacco BY-2 cells were transformed with a chimeric gene encoding an ER-targeted green fluorescent protein (30 kDa GFP-HDEL). In the transformed cells at the stationary phase, GFP fluorescence was observed in the vacuoles. A subcellular fractionation revealed that a trimmed form of 27 kDa GFP was localized in the vacuoles. Treatment with E-64d, an inhibitor of papain-type cysteine proteinases that inhibits the degradation of GFP in the vacuoles, resulted in a stable accumulation of 27 kDa GFP in the vacuoles, even in the logarithmic phase. Our results suggest that endogenous ER residents are transported constitutively to the vacuoles by bypassing the Golgi complex and are then degraded. [ABSTRACT FROM AUTHOR]

The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsrl mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 125 globulin in a Ca[SUP2+]-dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants. [ABSTRACT FROM AUTHOR]

Plants degrade cellular materials during senescence and under various stresses. We report that the precursors of two stress-inducible cysteine proteinases, RD21 and a vacuolar processing enzyme (VPE), are specifically accumulated in ~0.5 µm diameter × ~5 µm long bodies in Arabidopsis thaliana. Such bodies have previously been observed in Arabidopsis but their function was not known. They are surrounded with ribosomes and thus are assumed to be directly derived from the endoplasmic reticulum (ER). Therefore, we propose to call them the ER bodies. The ER bodies are observed specifically in the epidermal cells of healthy seedlings. These cells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal cells, the ER bodies start to fuse with each other and with the vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct pathway differs from the usual case in which proteinases are transported constitutively from the ER to the Golgi complex and then to vacuoles, with intervention of vesicle-transport machinery, such as a vacuolar-sorting receptor or a syntaxin of the SNARE family. Thus, the ER bodies appear to be a novel proteinase-storing system that assists in cell death under stressed conditions. [ABSTRACT FROM PUBLISHER]

We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments. [ABSTRACT FROM PUBLISHER]

The binding protein (BiP) has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of aberrant polypeptides. To elucidate the involvement of BiP in the biosynthesis of vacuolar proteins, we have characterized the protein in pumpkin cotyledons during seed maturation and seedling growth. Isolated microsomes from maturing pumpkin cotyledons contained a significant amount of BiP, protein-disulfide isomerase and calreticulin. We have purified a 70-kDa protein; sequences of the N-terminus and internal fragments of this protein exhibited a high identity to the sequence of soybean BiP. Immunoblot analysis with specific antibodies raised against the purified BiP showed that the amount of BiP in a cotyledon increased markedly at the middle stages and then decreased. The increase was accompanied by the synthesis of storage proteins and the development of the endoplasmic reticulum in the cotyledons at the middle stage of seed maturation. Most of these storage proteins degraded dramatically between 2 and 5 days after seed germination, and the degradation was also accompanied by a rapid increase in the level of BiP. Subcellular fractionation of the 4-day-old cotyledons showed a high accumulation of BiP in the endoplasmic reticulum. It is possible that BiP might be involved in the synthesis of seed storage proteins during maturation and in the synthesis of hydrolytic enzymes responsible for the degradation of the storage proteins during seed germination. [ABSTRACT FROM AUTHOR]