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During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane. [ABSTRACT FROM PUBLISHER]

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for AtERD2 with six transmembrane domains. [ABSTRACT FROM AUTHOR]

It is generally accepted that plant cells can contain multiple vacuoles with different functions, for example lytic vacuoles with lysosome-like properties and protein storage vacuoles for reserve accumulation. Recent data call into question the generality of this theory. In this study, we review the published evidence for the existence of multiple vacuoles. We conclude that the multivacuole hypothesis is valid for a number of cases, but care should be taken before assuming that it applies universally. [ABSTRACT FROM AUTHOR]

In developing pea cotyledons, storage proteins are sorted via dense vesicles into the protein storage vacuole. Formation of these unique transport vesicles is characterized by aggregation of their cargo proteins. Protein sorting into dense vesicles is pH dependent. In order to gain insight into the molecular basis of storage protein sorting, a membrane binding assay was developed which allows for a detailed biochemical analysis of binding events. Employing this assay it was possible to show that storage proteins bind in a pH-dependent manner to the membranes of the secretory pathway with a pH optimum in the range of the lumenal pH of the Golgi cisternae. Through reconstitution experiments, it was possible to demonstrate further that this recruitment occurs via the interaction of peripheral rather than intrinsic membrane proteins. Results of co-immunoprecipitation experiments point to interactions between different storage proteins in the secretory system. These results are discussed in terms of the aggregation-mediated sorting of storage proteins into maturing dense vesicles. [ABSTRACT FROM PUBLISHER]

The plant vacuolar sorting receptor (VSR) binds proteins carrying vacuolar sorting signals (VSS) of the ‘sequence-specific’ type (ssVSS) but not the C-terminal, hydrophobic sorting signals (ctVSS). Seeds of Arabidopsis mutants lacking the major VSR isoform, AtVSR1, secrete a proportion of the proteins destined to storage vacuoles. The sorting signals for these proteins are not well defined, but they do not seem to be of the ssVSS type. Here, we tested whether absence of VSR1 in seeds leads to secretion of reporter proteins carrying ssVSS but not ctVSS. Our results show that reporters carrying either ssVSS or ctVSS are equally secreted in the absence of VSR1. We discuss our findings in relation to the current model for vacuolar sorting. [ABSTRACT FROM AUTHOR]

Plant cells are considered to possess functionally different types of vacuoles in the same cell. One of the papers cited in support of this concept reported that protein storage and lytic vacuoles in root tips of barley (Hordeum vulgare) and pea (Pisum sativum) seedlings were initially separate compartments that later fused to form a central vacuole during cell elongation. We have reinvestigated the situation in these two roots using immunogold electron microscopy as well as immunofluorescence microscopy of histological sections. Using antisera generated against the whole protein of a-tonoplast intrinsic protein (TIP) as well as specific C-terminal TIP peptide antisera against α-, γ-, and δ-TIP, together with antisera against the storage proteins barley lectin and pea legumin and vicilin, we were unable to obtain evidence for separate vacuole populations. Instead, our observations point to the formation of a single type of vacuole in cells differentiating both proximally and distally from the root meristem. This is a hybrid-type vacuole containing storage proteins and having both α- and γ-TIPs, but not δ-TIP, in its tonoplast. As cells differentiate toward the zone of elongation, their vacuoles are characterized by increasing amounts of γ-TIP and decreasing amounts of α-TIP. [ABSTRACT FROM AUTHOR]

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV. [ABSTRACT FROM AUTHOR]

Receptors for acid hydrolases destined for the lytic compartment in yeast and mammalian cells are retrieved from intermediate, endosomal organelles with the help of a pentameric protein complex called the retromer. We cloned the Arabidopsis thaliana homologs of the three yeast proteins (Vps35, Vps29, and Vps26) constituting the larger subunit of retromer and prepared antisera against them. With these antibodies, we demonstrated the presence of a retromer-like protein complex in salt extracts prepared from Arabidopsis microsomes. This complex is associated with membranes that coequilibrate with prevacuolar compartment markers and with high-density sedimenting membranes. Immunogold negative staining identified these membranes as 90-nm-diameter coated microvesicles. Confocal laser scanning immunofluorescence studies performed on tobacco (Nicotiana tabacum) BY-2 cells revealed high degrees of colabeling between all three retromer antisera and the prevacuolar compartment (PVC) markers PEP12 and vacuolar sorting receptor VSRAt-1. The presence of plant retromer at the surface of multivesicular bodies was also demonstrated by immunogold labeling of sections obtained from high-pressure frozen/freeze-substituted specimens. Treatment of BY-2 cells with wortmannin led to swelling of the PVC and a separation of the VPS35 and VSR signals. Preliminary data suggesting that retromer interacts with the cytosolic domain of a VSR were obtained by immunoprecipitation experiments performed on detergent-solubilized microsomes with Vps35 antibodies. [ABSTRACT FROM AUTHOR]

The vacuole of plant cells is no longer considered to be a single compartment with multifunctional properties. A lot of evidence now points to the presence of multiple functionally distinct vacuolar compartments, some existing side by side in the same cell. As a consequence, the plant Golgi apparatus is faced with the problem of recognizing proteins destined for lytic and storage vacuoles and segregating them individually from the flow of secretory proteins to the cell surface. In contrast to acid hydrolases, which are sorted by BP-80-like receptors at the trans-Golgi of plant cells, the identification of receptors for storage proteins has in many ways resembled ‘the search for the Holy Grail’. There are several candidates for storage protein receptors, but in no single case is the evidence entirely convincing. Much of the problem lies in the lack of consensus, sorting sequences in the proteins investigated. Other difficulties stem from ‘out-of-context’ heterologous expression studies. Evidence is now accumulating for the participation of hydrophobic sequences in inducing the formation of protein aggregates in the early Golgi apparatus, for which classical sorting receptors do not appear to be necessary. This review critically examines the current situation and contrasts the differences between data obtained in situ and data obtained transgenically. It highlights the so-called ‘dense-vesicle’ pathway and culminates with a discussion on the hitherto neglected problem of the intracellular transport of storage protein processing enzymes. [ABSTRACT FROM AUTHOR]

Developing pea seeds contain two functionally distinct vacuoles– lytic vacuoles and protein storage vacuoles (PSV). The Golgi apparatus of these cells has to discriminate between proteins destined for these vacuolar compartments. Whereas it is known that sorting into the lytic vacuole is performed via the conserved clathrin-coated vesicle pathway, sorting of proteins into the protein storage vacuole remains enigmatic. In developing pea cotyledons, the major storage proteins are sorted via‘dense vesicles’. In this report we examined the sorting of a minor protein of the protein storage vacuole, the sucrose-binding-protein homolog (SBP), along the secretory pathway employing immunoelectron microscopy on cryosectioned pea cotyledons. SBP follows the same vesicular route into the PSV as the main storage proteins legumin and vicilin, via the dense-vesicles. Furthermore, legumin and SBP are sorted together into the same dense vesicle population at the stack. Although soluble cargo proteins of the dense vesicles, they show a stratified distribution in the lumen of the dense vesicles. Whereas the legumin label is equally distributed across the lumen, the SBP label is concentrated at the membrane of the vesicle. This observation is discussed with respect to a putative receptor-mediated sorting of the proteins into the dense vesicles. [ABSTRACT FROM AUTHOR]

Secretion, endocytosis and transport to the lytic compartment are fundamental, highly coordinated features of the eukaryotic cell. These intracellular transport processes are facilitated by vesicles, many of which are small (100 nm or less in diameter) and ‘coated’ on their cytoplasmic surface. Research into the structure of the coat proteins and how they interact with the components of the vesicle membrane to ensure the selective packaging of the cargo molecules and their correct targeting, has been quite extensive in mammalian and yeast cell biology. By contrast, our knowledge of the corresponding types of transport vesicles in plant cells is limited. Nevertheless, the available data indicate that a considerable homology between plant and non-plant coat polypeptides exists, and it is also suggestive of a certain similarity in the mechanisms underlying targeting in all eukaryotes. In this article we shall concentrate on three major types of transport vesicles: clathrin-coated vesicles, COP-coated vesicles, and ‘dense’ vesicles, the latter of which are responsible for the transport of vacuolar storage proteins in maturing legume cotyledons. For each we will summarize the current literature on animal and yeast cells, and then present the relevant data derived from work on plant cells. In addition, we briefly review the evidence in support of the ‘SNARE’ hypothesis, which explains how vesicles find and fuse with their target membrane. [ABSTRACT FROM AUTHOR]

A fraction enriched in endoplasmic reticulum and Golgi membranes from developing cotyledons of Pisum sativum L. has proved to be a convenient source for the isolation of prolegumin, the precursor of the major 11S storage globulin of pea seeds. Two pro-proteins were isolated with molecular masses of 60 kDa and 75 kDa, respectively. A monoclonal antibody, designated 2B1, against prolegumin was raised using the in vitro immunization technique. This antibody recognizes the 60 kDa precursor polypeptide, but only the 20 kDa β-subunit of mature legumin. Prolegumin, like the β-subunit of the mature legumin, is a hydrophobic protein. After import into the protein storage vacuole, and after formation of the protein bodies trimeric 9S prolegumin assembles into 12S hexamers without prior processing of the precursor. Since prolegumin in vitro does not oligomerize into more than 9S tnmers these results suggest that a protein-mediated assembly of 9S prolegumin trimers into 12S prolegumin hexamers probably occurs in the lumen of the protein storage vacuole. Prolegumin, but not mature legumin, binds very tightly to membranes. This property points to a possible way of identifying a putative prolegumin receptor. [ABSTRACT FROM PUBLISHER]

In this report we demonstrate that a 51-kDa outer-envelope membrane protein (P51) is involved in protein translocation into chloroplasts. Furthermore it is shown that phosphorylation of P51 is functionally related to the process of binding and/or importing precursor proteins into chloroplasts. Several lines of evidence have been obtained supporting this suggestion. First, protein import into chloroplasts was inhibited by the membraneimpermeable agent pyridoxal Y-phosphate, which has been shown to react with a component of the proteinimport apparatus. Phosphorylation of envelope membrane polypeptides using [γ-32P]ATP in the presence of pyridoxal 5'-phosphate resulted in an increased incorporation of 32P radiolabel into a 51-kDa membrane polypeptide (P51). A close correlation between the inhibition of protein import and the increase in the phosphorylation state of P51, both as a function of PLP concentration, was observed. Second, binding of purified precursor proteins to chloroplasts resulted in a specific increase in the phosphorylation state of P51. This effect was not exerted by the mature form of the precursor protein lacking the presequence. Third, internally generated ATP was able to compete specifically with externally added [γ-32P]ATP for the phosphorylation of P51. Fourth, digestion of the outer-envelope membrane with low amounts of thermolysin resulted in a loss of protein import activity, which was associated with the removal of the phosphorylation site of P51. Phosphorylation of P51 proceeds with an apparent Km (ATP) of about 5 µM, which is much lower than the ATP concentration required for the protein translocation itself. We suggest that two different ATP-dependent processes are involved in protein translocation into chloroplasts. P51 represent presumably a regulatory component of the protein-import apparatus or the protein receptor itself. [ABSTRACT FROM AUTHOR]

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast. [ABSTRACT FROM AUTHOR]

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast. [ABSTRACT FROM AUTHOR]

The tonoplast is usually characterized by the presence of two electrogenic proton pumps: a vacuolartype H-ATPase and a pyrophosphatase, as well as a putative water-channel-forming protein (γ-TIP). Using a post-embedding immunogold labelling technique, we have detected the presence of these transport-protein complexes not only in the tonoplast, but also in the plasma membrane and trans Golgi elements of maturing pea ( Pisum sativum L.) cotyledons. These ultrastructural observations are supported by Western blotting with highly purified plasma-membrane fractions. In contrast to the vacuolar-type H-ATPase, whose activity was not measurable, considerable pyrophosphatase activity was detected in the plasma-membrane fraction. These results are discussed in terms of a possible temporary repository for tonoplast proteins en route to the vacuole. [ABSTRACT FROM AUTHOR]