Giselbert Hinz

Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the golgi apparatus of developing pea cotyledons in different transport vesicles

In the parenchyma cells of developing legume cotyledons, storage proteins are deposited in a special type of vacuole, known as the protein storage vacuole (PSV). Storage proteins are synthesized at the endoplasmic reticulum and pass through the Golgi apparatus. In contrast to lysosomal acid hydrolases, storage proteins exit the Golgi apparatus in 130-nm-diameter electron-dense vesicles rather than in clathrin-coated vesicles. By combining isopycnic and rate zonal sucrose density gradient centrifugation with phase partitioning, we obtained a highly enriched dense vesicle fraction. This fraction contained prolegumin, which is the precursor of one of the major storage proteins. In dense vesicles, prolegumin occurred in a more aggregated form than it did in the endoplasmic reticulum. The putative vacuolar sorting receptor BP-80 was highly enriched in purified clathrin-coated vesicles, which, in turn, did not contain prolegumin. The amount of BP-80 was markedly reduced in the dense vesicle fraction. This result was confirmed by quantitative immunogold labeling of cryosections of pea cotyledons: whereas antibodies raised against BP-80 significantly labeled the Golgi stacks, labeling of the dense vesicles could not be detected. In contrast, 90% of the dense vesicles were labeled with antibodies raised against α-TIP (for tonoplast intrinsic protein), which is the aquaporin specific for the membrane of the PSV. These results lead to the conclusions that storage proteins and α-TIP are delivered via the same vesicular pathway into the PSVs and that the dense vesicles that carry these proteins in turn do not contain BP-80.

The molecular characterization of transport vesicles

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.

Immunological detection of tonoplast polypeptides in the plasma membrane of pea cotyledons

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.

Vesicle transfer of storage proteins to the vacuole: The role of the Golgi apparatus and multivesicular bodies

Summary The storage proteins vicilin and legumin pass through the Golgi apparatus (GApp) on their way to the protein storage vacuole (PSV). Upon entering the GApp vicilin and legumin are efficiendy segregated to the periphery of the cisternae where they begin to assemble into electron-dense aggregates. These form the nuclei of so-called dense vesicles (DV), which, presumably by cisternal progression through the stack, eventually reach the trans face of the GApp. By this time their contents have become even more condensed and many have obtained a partial clathrin coat on their cytosolic surface. Clathrin coated vesicles (CCV), which appear to bud from the DV may represent a mechanism for the retrieval of proteins missorted into the DV. The DV do not transfer their contents direct to the PSV. Instead, as is the case in yeasts and mammalian cells, an intermediate compartment is implicated. In pea cotyledon cells this takes the form of large (at least 1 μm diameter), multivesicular bodies (MVB). Double immunogold labeling with globulin antisera suggests that the MVB fuse with the PSV leading to a stratification of storage proteins in deposits at the tonoplast. Experiments with monensin lead to a depletion of the contents of the MVB and a redirection of globulin deposition to the cell surface. CCV, DV and MVB have been probed with antibodies against the vacuolar sorting receptor BP-80. CCV and MVB react positively, whereas there is no signal with the DV.

VACUOLE BIOGENESIS AND PROTEIN TRANSPORT TO THE PLANT VACUOLE : A COMPARISON WITH THE YEAST VACUOLE AND THE MAMMALIAN LYSOSOME

SummaryThe vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of “vacuolar proteins” within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.

One Vacuole or two Vacuoles: Do Protein Storage Vacuoles Arise de novo during Pea Cotyledon Development?

Using improved fixation procedures for both conventional and immuno-electron microscopy, we have reinvestigated the problem of protein storage vacuole (PSV) biogenesis in developing pea (Pisum sativum L.) cotyledons. Our results show that PSV develop de novo and the vegetative vacuoles degenerate. The PSV appear to arise from the rough ER as judged by the presence of osmiophilic substances in the lumen of rER cisternae and by the detection of a BiP analogue in the storage protein deposits contained in the young PSV. These results are discussed in terms of the involvement of other endomembrane compartments (Golgi apparatus and clathrin coated vesicles) in the intracellular transport of storage proteins in legumes

Localization of pyrophosphatase in membranes of cauliflower inflorescence cells

Abstract. Using a polyclonal antiserum specific for the tonoplastic H+-pyrophosphatase (tPPase), significant amounts of antigenic polypeptides of the correct molecular mass were detected in Western blots of plasma membrane isolated from cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning and subsequent sucrose density centrifugation. Potassium iodide-stripped plasma membranes continued to give a strong positive signal, indicating that the PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained negligible vacuolar (v)H+-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a proton-pumping activity with ATP as substrate, but such an activity was not measurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast-containing fractions. Immunogold electron microscopy confirmed the presence of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was higher at the plasma membrane than at the tonoplast, except for membrane fragments occurring in the lumen of the vacuoles which stained very conspicuously.

Golgi-mediated Transport of Seed Storage Proteins

The great majority of seed proteins that are stored in the vacuole prior to desiccation are transported via the Golgi apparatus. In this organelle they are separated from other products of the secretory pathway. Evidence is accumulating that the mechanism for segregation of storage proteins is different from that of soluble proteins destined for lytic vacuoles: it rarely seems to require short targeting propeptides at the N- or C-terminus. Instead, the three-dimensional conformation of the protein appears to be a critical factor, leading to self-assembly into osmiophilic aggregates. Also unusual is that this process starts immediately after entry into the Golgi apparatus, i.e. at the cis -cisternae, rather than at the trans -pole where acid hydrolases are packaged into clathrin-coated vesicles. Storage protein aggregates accumulate into so-called “dense” vesicles at the periphery of the cisternae and are transported towards the trans -pole of the Golgi apparatus by cisternal progression. Before the dense vesicles are released, clathrin-coated vesicles form at their surface; however, the function of the latter remains the object of speculation. In other eukaryotes, delivery of Golgi-derived lumenal products to the vacuole does not occur directly, but via a pre-vacuolar compartment. There is evidence that this is also the case for plants, and in developing pea cotyledons the pre-vacuolar compartment takes the form of a large multivesicular body. Ultimately this appears to fuse in toto with the protein storage vacuole.

Ultrastructure of the pea cotyledon Golgi apparatus: Origin of dense vesicles and the action of brefeldin A

SummaryWe have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, “dense” vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborgs medium for 5 h without the inhibitor.

Stratification of Storage Proteins in the Protein Storage Vacuole of Developing Cotyledons of Pisum sativum L.

Summary Legumin, vicilin and pea albumin are the most prominent storage proteins in pea seeds. In order to localize these proteins in the developing cotyledons we have employed an improved fixation procedure for immunogold labelling for electron microscopy. Profiles of protein bodies (PB, 1-2 µm diameter) label heavily and specifically with antibodies against vicilin and legumin. By contrast, such PB are only weakly labelled with antibodies against pea albumin 1. Antibodies against pea albumin 1 label a narrow, granulated zone at the luminal borders of the deposits. Depending on their size, storage protein deposits at the tonoplast of the protein storage vacuole (PSV) show different labelling patterns: small deposits label positively only with vicilin antibodies; labelling with legumin is seen in large deposits but is restricted to the tonoplast rather than luminal borders of the deposits. We frequently observed profiles of storage protein deposits at the tonoplast of the PSV, which were elongated and protruded into the cytoplasm. A gradient of legumin labelling in these deposits was seen. We interpret such images as stages in the formation of PB, which apparently arise by budding at the surface of the PSV.