Endoplasmic Reticulum to Vacuole Trafficking of Endoplasmic Reticulum Bodies Provides an Alternate Pathway for Protein Transfer to the Vacuole

A, Shown are OBs and PBs of a late maturation soybean cotyledon from a transgenic plant that suppresses conglycinin, one of the two major storage proteins. A large fraction of the other storage protein glycinin is diverted from endomembrane progression and forms ER-derived protein bodies. Both the OBs and PBs have ribosomes associated with the surface that is a remnant of the ER origin of both of these organelles. See Kinney et al. (2001) for details. B, Autophagic vesicles in the PSV of mung bean cotyledons after germination is shown. The autophagic vesicles contain fragmented remnants of the ER network that remains from seed maturation and is degraded postgermination. See Herman et al. (1981) for details. C, Rubber particles (RP) in the cytoplasm and taken in the vacuole by autophagy in Guayule (Parthenium argentatum) are shown. Although ER-derived, the rubber particles do not possess bound ribosomes like PBs and some OBs. See Backhaus and Walsh (1983) for details. D, The autophagy of zein PBs into the PSV of transgenic tobacco seed cells is shown. Zein PBs are initially assembled by the ER and then subsequently sequestered into the protein storage vacuole by selective autophagy. While autophagy of the zein PBs is occurring, other cellular structures and organelles are not subjected to autophagy. This separates the selective autophagy of PBs from the nonspecific autophagy that is central to programmed cell death and senescence. See Coleman et al. (1996) for details. The plant ER is a large network of interconnected sheets and tubes (Staehelin, 1997) contiguous with the nuclear envelope and exists as a patchwork of specialized domains that are differentiated with respect to the ability of ER to synthesize proteins and other substances (Staehelin, 1997; Choi et al., 2000). The specialization of ER domains provides the functional capacity to compartmentalize the synthesis of specific proteins and glycoproteins and other substances synthesized and processed by the ER, such as lipids, oil, waxes, rubber, sterols, and isoprenoids regulated by development, stress, and environmental conditions. While there is still a great deal to be learned about how various domains and/or components of the ER are used by plants to produce specific components, there is considerable data that supports the concept of specialized ER subdomains with specific functions. Autophagy in plants appears to be a continuous process in most cells that is illustrated by the presence of partially degraded material in the vacuoles of cells in most tissues. Although first detailed a few decades ago (e.g. Matile, 1975), much remains to be elucidated on autophagy's regulation and function. The amount of apparent autophagy is greatly enhanced during cellular remodeling events that occurs in development, in reaction to stress and recovery, and during senescence. For example, Suc starvation induces autophagy in cultured cells to recover carbon and nitrogen resources to maintain life under nutrient-challenged circumstances (Moriyasu and Ohsumi, 1996). Autophagy also appears to have a significant role in renewal and turnover of the tonoplast (Melroy and Herman, 1991). The tonoplast is variable in composition with major compositional changes occurring during development and in response to stress. Autophagy in plants results from the engulfment of cytoplasmic materials by the tonoplast, resulting in formation of a vesicle within the vacuole sequestering the engulfed material (for review, see Herman, 1994). For membranous organelles this results in the appearance of a double unit membrane of the engulfed material with the inner membrane being that of the organelle and the outer membrane being that of the engulfing tonoplast. Once isolated within the vacuole the action of vacuolar hydrolases lyses the outer former tonoplast membrane, exposing the sequestered material to the vacuolar hydrolases that include proteases, glycosidases, phosphatases, nucleases, and phospholipases that then degrade the inner organelle membrane and its contents. How cytoplasmic material is selectively identified for autophagy remains unknown and an important subject for future research. The engulfment of cytoplasmic material by the tonoplast in plants differs from the autophagic process observed in animals and yeast. Although the processes are superficially similar, ending with deposition in the lytic compartment, the process of sequestration into the lytic compartment is different. In yeast and animals, cytoplasmic material is engulfed in membrane that is either de novo synthesized or is Golgi-derived and subsequently is recruited and encases the material targeted for autophagy. This results in a cytoplasmic structure with a double unit membrane. The outer membrane fuses with the lysosome (animals) or vacuole (yeast), releasing the sequestered organelle into the lytic compartment for degradation (for review, see Klionsky and Ohsumi, 1999). Because the ER has specialized functions, ER membrane turnover is necessary to dispose of specialized ER inventory and/or redundant components by changing developmental or environmental circumstances. There are few studies on the disposal of ER membranes. The role of the vacuole to use autophagy to sequester and degrade the ER was established by observations on postgermination mung beans (Vigna radiate; Herman et al., 1981). The mung bean cotyledon ER undergoes massive proliferation during seed maturation to produce storage proteins, and on desiccation this ER network is fragmented but remains in the dormant seed (Gilkes et al., 1979). Immediately after germination this ER is engulfed by the protein storage vacuoles (PSVs) and degraded while a de novo synthesized ER network is formed to support the mobilization of seed storage substances (Fig. 1B). Therefore, in at least some circumstances there is selective autophagy of ER by the vacuole for disposal. The vacuolar autophagy of ER bodies is a variation on this autophagic destruction of redundant ER. The first unambiguous demonstration of an ER body trafficking to the vacuole is sequestration of PBs containing hydrophobic prolamin proteins in wheat (Triticum aestivum; Levanony et al., 1992). Wheat prolamins are initially assembled as protein accretions in the ER that are then budded from the ER as PBs with a rough ER-limiting membrane. Electron microscopy shows that the PBs are engulfed protovacuoles by autophagy. As the small vacuoles fuse the PB is left encased in the enlarging vacuole. This is then a variation on the theme of normal vacuole ontogeny in which prevacuoles bud from the Golgi, and fuse one to another, gradually enlarging the protovacuole forming the large central vacuole. The ER-derived limiting membrane is lysed, releasing the prolamin storage protein accretions that are apparently resistant to the action of the vacuolar hydrolases. The prolamin accretions merge in parallel with the enlargement of the vacuole, resulting in a large central vacuole of the endosperm with one or more large prolamin accretions at the end of seed maturation. Wheat endosperm, like that of other monocotyledonous plants, dies at the end of maturation, leaving the prolamin accretions within the dead endosperm. Not all prolamin PBs are targeted for vacuolar sequestration. Maize, for example, possesses zein-containing ER-PBs that at least in part remain contiguous with the ER from which they were synthesized or are free in the cytoplasm (for review, see Herman and Larkins, 1999). Curiously, replicating the process of zein-PB assembly in transgenic tobacco (Nicotiana tabacum) seeds results in the formation of PBs containing zein accretions surrounded by an ER-derived ribosome-studded membrane, but these PBs are subject to autophagy by tobacco seed PSVs (Coleman et al., 1996; Fig. 1D). A similar process is the sequestration of rubber in the vacuole. Rubber particles are ER bodies containing a central core with an accretion of ER-synthesized high-Mr rubber (Backhaus and Walsh, 1983; Fig. 1C). Rubber particles bud from the ER, but unlike PBs with a ribosome studded membrane, rubber particles possess a membrane that primarily consists of a protein that is a member of the Cytochrome P450 family (Pan et al., 1995). After sequestration in the vacuole the rubber membrane or coat is digested, leaving intravacuolar naked rubber particles that self-aggregate, forming large rubber complexes as occurs with the wheat prolamin PB cores. Hydrophilic storage proteins of the 2, 7, and 11S families are conserved and widely distributed in plant seeds. These proteins are all synthesized by the ER and are largely deposited in the PSVs by endomembrane progression through the Golgi. Targeting sequences have been identified for many of these proteins that are recognized by a Golgi receptor, and in some instances glycan side chains of the precursor storage proteins are modified by Golgi enzymes, further demonstrating an endomembrane progression path for deposition. Yet, Hara-Nishimura and co-workers observed that precursors of pumpkin (Cucurbita pepo) 2S storage proteins are also found in small dense vesicles surrounded by a rough ER membrane (Hara-Nishimura et al., 1998). Unlike prolamins that self-aggregate to produce PBs, these globulins are hydrophilic proteins that were previously all assumed to be deposited in vacuoles by endomembrane progression through the Golgi. There were prior hints that hydrophilic globular storage proteins might aggregate in the ER, producing PBs by expressing proteins such as 7S vicilin with an ER-retention sequence K/HDEL (Wandelt et al., 1992), but this is an artificial transgenic consequence. Storage globulins normally have a relatively short T1/2 in the ER lumen before progressing to the Golgi, but greatly increasing the ER residence time promotes the self-aggregation of the protein leading to self-assembly of PBs. There are now other examples of this phenomenon that extend to other foreign proteins expressed in transgenic plant cells. But the Hara-Nishimura et al. (1998) experiments showed that a phenomenon that had previously been known only as the consequence of expressing transgenes also occurred as a naturally occurring mechanism for storage protein accumulation. In their model the pro2S storage protein is aggregated in the ER and budded from the ER in PBs (termed by them as PAC vesicles) that are sequestered in the vacuole, where the pro2S protein is processed to its mature form (Hara-Nishimura et al., 1998). Whether the transfer into the vacuole is accomplished by vacuolar engulfment autophagy remains to be demonstrated. The pumpkin 2S protein fused to phosphoinothricin acetyltransferase induces protein body formation as a transgene expressed in Arabidopsis (Arabidopsis thaliana; Hayashi et al., 1999). The 2S storage protein family is distributed among diverse plant species. Hence, the pumpkin 2S globulin results suggest that there are likely many other examples of both endomembrane progression and ERvt paths for storage protein accumulation to be identified. What would be of particular interest is to investigate whether the same seeds use both mechanisms, and if so whether there is a process of regulating the choice of pathway either by developmental stage or environmental circumstance or other biological factors. Other storage proteins of the 7S (vicilin-type) and 11S (legumin-type) have been shown to be synthesized by the ER, form oligomers, and are then transported to the vacuole by endomembrane progression through the Golgi. However, suppression of the soybean (Glycine max) 7S conglycinin storage protein by silencing results in seeds that accumulate 11S storage protein that has been substituted for the missing 7S protein. The pro11S protein glycinin forms accretions in the ER, forming PBs that accumulate during seed maturation (Fig. 1A; Kinney et al., 2001). The accumulated inventory of PBs is present in the fully mature soybeans. The 11S PBs are eventually engulfed by the vacuole by autophagy, but this does not occur until after seed germination. Storage proteins are also found in vegetative cells including leaves, stems, tubers, and pollen and are different genes and gene families than the seed storage proteins. Although it is widely assumed these proteins are deposited in the vacuole by endomembrane progression through the Golgi, in most instances there is little experimental evidence to prove this. It is possible that PBs and the ERvt pathway may have some role in nonseed storage protein accumulation, and this needs to be a subject of future investigations. Vegetative cells are often subjected to wide variations of environmental conditions, and it is possible that the endomembrane progression and ER body formation occurs simultaneously or under specific circumstances as occurs in transgenic soybean seeds lacking α-conglycinin (Kinney et al., 2001). The discovery that KDEL tailed-Cys protease precursors (Tanaka et al., 1991) form into the core of ER bodies termed protease precursor vesicles (PPV; Schmid et al., 1998; Toyooka et al., 2000; for review, see Chrispeels and Herman, 2001) is a remarkable adaptation by plants and as far is presently known plant-specific. The Cys protease papain superfamily is a large diverse family of enzymes of ancient origin found in most if not all eukaryotes. Cys proteases are initially synthesized as inactive precursors by the ER with a large prodomain that lies in the groove containing the active site precluding proteolytic activity until the enzymes are activated at their destination site. In general, the proteases exit the ER, are transported to and through the Golgi where, depending on the targeting information, they are then transported to the cell's lytic compartment, lysosomes in animals, vacuoles in plants, or are secreted into extracellular space. The plant papain superfamily is large with individual members used in developmentally specific as well as general proteolytic functions involving both intracellular and extracellular protein degradation. The PPVs are assembled in cells undergoing remodeling and senescence, with storage protein mobilization and programmed cell death as examples (Schmid et al., 1998, 2001; Toyooka et al., 2000; Hayashi et al., 2001). That PPVs have been found to be associated with these two processes suggests one possible role for Golgi-bypass of Cys protease precursors. By forming PPVs the plant cell has the potential to accumulate a large inventory of protease in a stored, stable precursor form that is only activated when autophagy of the PPVs is initiated. By this means, instead of gradual changes in protein composition as the proteases are accumulated and function, this would instead confer the ability to mediate rapid changes that could be useful in storage protein mobilization and programmed cell death. For what are normally soluble monomeric precursors of Cys proteases to be sequestered into ER bodies requires that the protease precursors be separated from the bulk of secretory and ER-lumen proteins aggregated and marshaled into the nascent PPV ER body. This probably requires that the proteins undergo self-aggregation to provide a self-assembly mechanism to separate the aggregating protein from all of the other proteins present in the ER lumen. One approach to promote self-aggregation is to utilize the ER-retention system. ER-lumen proteins possess a carboxy-terminal retention sequence, H/KDEL, and some other variants such as KEEL that function in identifying the ER-lumen proteins for retrieval from the bulk protein flow at the cis-Golgi. Biologists working with plants, animal cells, and yeast have discovered that placing the ER-retention sequence on some secretory proteins results in these proteins being retained and/or retarded in the ER. In many, but not all, instances the normally soluble proteins aggregate as the result of engineering an ER-retention sequence onto the protein (Wandelt et al., 1992). These experiments anticipated the discovery of the aggregation of KDEL-containing Cys proteases (Schmid et al., 1998, 2001; Toyooka et al., 2000) in some plants to form precursor protease vesicles, a form of ER body. How the repeated cycles of ER-Golgi trafficking promote the self-aggregation of some proteins is unknown, but because this occurs with only some proteins it is likely an intrinsic property of those proteins. However, the KDEL sequence may not be absolutely essential to divert enzyme precursors into ER bodies. The presence of vacuolar an protease that functions to vacuolar precursor proteins to mature hydrolases and storage proteins, has been discovered in ER bodies et al., it possible for components of PPV to include other protease precursors that do not possess the ER-retention It is likely secretory protein precursor could be an protein accretion if it could be to with zein in that ER body formation is a self-assembly process et al., that protein that is to be in the ER and formed into accretions could then result in the of events to form an ER body and subsequently be deposited in the vacuole by autophagy. It is likely that further examples of precursor proteins aggregating in the ER be of the present evidence that the ER bodies from the ER are organelles. The presence of ribosomes on PBs, PAC vesicles, and PPVs suggests the potential that further protein could be to the organelles after from the ER network has Whether this would be with or by could then be if these organelles in being However, experiments to in sequestered on member ER body family or rough have not been or It would be of considerable interest to see if the only the prolamin storage protein core of the protein body or whether there are other not previously proteins that could be in further maturation ER body. The pathway of ER bodies ending with the autophagic sequestration in the vacuole an ERvt pathway that has parallel functions to the pathway in yeast and that also apparently exists in animal cells. It is useful to how this ERvt pathway and differs from the In yeast where the autophagic vesicle is assembled de it is necessary for the protein to membrane to the protein aggregate and subsequently to mediate its with the (for review, see and Ohsumi, 1996). This is accomplished by a of events where the protein is and bound to other proteins and then by membrane for the cytoplasmic assembly of the functions to transfer cytoplasmic synthesized to the vacuole et al., The of the gene to that in forms and This and proteins that the producing a The with protein that induces the to and be associated with a de novo synthesized sequestering vesicle membrane that encases the resulting in the formation of a body with a double unit membrane. The outer membrane of body then fuses with the vacuole, releasing a unit vesicle sequestering the into the vacuole where the membrane is by vacuolar releasing the for into a mature For structures the processes are to where organelles and large structures are encased in a membrane, which in fuses with the releasing the encased structure into the vacuole where it is subject to the action of lytic hydrolases. By one mechanism of proteins and structures in a membrane to mediate sequestration into the lytic compartment, yeast and animal cells have a conserved mechanism that processes of lytic enzyme composition of the vacuole in parallel with protein turnover and organelle disposal. There is evidence for ER trafficking the Golgi to the lysosome in animal cells. A that results from the synthesis of and is associated with a The protein in the ER, and the ER containing the protein is in and 2000). The ER to lysosome transfer occurs by a mechanism where the ER is encased in a de novo membrane and the is to the lytic compartment by of the membrane than engulfment and sequestration and 2000). The function of pathway is a of protein such as and In some instances these are by that proteins to be expressed with producing and aggregated proteins. Although the accumulation of these proteins is to a fraction of the protein may be from the cells by transfer to the lytic compartment by the et al., and within for It is that protein aggregation result in forming protein accretions that are targeted for sequestration in the lytic Although this is an parallel process to the ERvt path of ER bodies in plants, the is different. While of biological processes occur are often it is useful to what the ERvt and do provide the capacity of endomembrane progression through the Golgi. protein and rubber by the ERvt pathway provides a to possible on through the endomembrane system. For endomembrane proteins are in with their synthesis and from organelle to organelle targeting and occurs during and for many of the vacuolar and secreted enzymes and occurs after at the The vacuoles of plants and yeast have not been shown to possess protein deposited after by through a membrane as occurs in and This that all the proteins within the vacuole be and taken into the vacuole by there are two major of trafficking proteins to the vacuole. One is endomembrane progression where synthesized and proteins are transported to and through the Golgi and then to the vacuole. targeting to the vacuole requires targeting that is by Golgi with sequences on the vacuole protein. of the proteins sequestered in the vacuole are large oligomers, seed storage proteins, of or There may be of of protein for endomembrane progression from the ER, to and through the Golgi, and deposition of vacuole. The might that possible for endomembrane protein complexes to be transported to the vacuole that would be endomembrane progression through the Golgi. The ERvt pathway in have much capacity than the similar Because pathway requires specific with proteins that mediate formation of the and it is likely that these are membrane material to form the and into the vacuole is not In for the ERvt that might in the ER by self-assembly result in the formation of an ER body that be taken into the vacuole by autophagy. Although the only forms of ER bodies now known to be sequestered in the vacuole by ERvt are those containing either protein or rubber, this still a diverse of hydrophobic and hydrophilic storage proteins and enzyme precursors that is much than that for the The of plant cell is on a small of plants, primarily plants and a few model plants, most often under and for major vegetative and studies on plants, of plants, as well as of conditions, likely other examples of ER bodies and other proteins and other The potential of ER bodies to sequester ER-synthesized material and that material in vacuoles is only now being recognized as an important of plant cell A of microscopy observations the likely result in many other examples of being identified and on the development and environmental of a time when microscopy are because of of for there are to microscopy with other forms of cell and