Compartmentation of plant cell proteins in separate lytic and protein storage vacuoles

Compartmentation of plant cell proteins in separate lytic and protein storage vacuoles

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© 1998 by Gustav Fischer Verlag, Jena

Compartmentation of Plant Cell Proteins in Separate Lytic and Protein Storage Vacuoles





Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA Received June 26, 1997 . Accepted October 30, 1997


Plant cells may contain separate protein storage and lytic vacuolar compartments. Two separate pathways carry soluble proteins from the Golgi to the appropriate vacuolar compartment. A novel protein family, the Vacuolar Soning Receptor family, is involved in sorting proteins to the lytic vacuole via clathrin CQated vesicles. Characteristics of these VSR proteins, and evidence that they traffic between Golgi and a prevacuolar compartment are reviewed.

Key words: Pisum sativum, Arabidopsis thaliana, akurain, clathrin, receptor, vesicle traffic. Abbreviations: CCV = clathrin coated vesicle; LV = lytic vacuole; NPIR =Asn-Pro-Ile-Arg; PSV = protein storage vacuole; SOV =smooth dense vesicle; TIP = tonoplast intrinsic protein; VSR =vacuolar sorting receptor. Introduction

The plant cell vacuole has long been considered to be a multifunctional compartment (Boller and Wiemken, 1986; Wink, 1993). For example, vacuoles may have features similar to those of lysosomes, and contain hydrolytic enzymes such as glycosidases and proteases that function in an acidic pH environment (Boller and Kende, 1979; Matile, 1975). Vacuoles may concentrate organic acids, anthocyanins, and a diverse population of secondary metabolic products including alkaloids, glycosides, and glutathione conjugates that may be toxic to predators or to the plant itself (Boller and Wiemken, 1986; Marrs et al., 1995; Wink, 1993). Another, agriculturally imponant, function for plant vacuoles is to store proteins. Although the specialized process by which proteins are stored in protein bodies during seed development (Higgins, 1984) has been studied in most detail, transient storage of proteins in vacuoles may occur in a wide range of cell types (e.g. (Greenwood et al., 1986; Herman, 1994; Herman et al., 1987; Staswick, 1994). Protein storage vacuoles in seeds were thought to originate by subdivision of the central vacuole as storage proteins were deposited and formed protein bodies (Craig et al., 1979; Craig et al., 1980; Vitale and Chrispeels, 1984). This apparj. Plant Physiol. Wli. 152. pp. 653-658 (1998)

ent multifuncrional propeny of a single plant vacuole posed contradictions that were not addressed. Specifically, it was difficult to understand how a diverse array of storage proteins could enter an acidic compartment with acrive proteases and survive intact to form protein bodies. The contradiction was emphasized by the knowledge that, after germination, protein bodies apparendy fused and became incorporated within vacuoles where active degradation of the storage proteins took place (Nishimura and Beevers, 1978, 1979). Similarly, there were apparendy contradictory results from studies addressing the biogenesis of vacuoles. It was apparent that the morphology of vacuoles within a single cell type could change drastically during cell development or with changes in metabolic activity (Campbell and Garber, 1980; Herman et al., 1994; Marty et al., 1980; Palevitz et al., 1981). Some morphologic studies indicated that vacuoles originate from a Golgi-associated tubular membranous network that could enlarge in part by autophagic incorporation of ponions of the cytoplasm (Marty, 1978; Marty et al., 1980). Alternatively, it was suggested that vacuoles originated direcdy from endoplasmic reticulum (Burgess and Lawrence, 1985; Hilling and Amelunxen, 1985). Recent studies have provided new insights into these some of these problems. Specifically, it is now clear that funcrion-





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aleurain, a barley cysteine protease, first indicated to us that two vacuolar compartments might exist in some cells. Antibodies were raised to a recombinant fusion protein containing the protease domain of aleurain, and affinity purified. When used in immunogold electron microscopy (EM) stud• ies of aleurone layers from imbibed seeds that had not yet germinated, antibody labeling was found exclusively over +moderately electron dense vacuoles that we termed «aleuraincontaining vacuoles». Interestingly, the antibodies did not label protein storage vacuoles (PSVs) that contained protein bodies and other inclusions such as phytic acid globoid (Hol~1 • werda et al., 1990). In subsequent work, PSVs were purified .• s~. from barley aleurone protoplasts by Bethke et al. (1996); consistent with our earlier results, these isolated PSVs did not contain immunologically detectable aleurain (R. L. Jones, pers. comm.). Knowledge of the steps involved in aleurain biosynthesis a-TIP plus functional characterization of the enzyme strongly support the concept that the aleurain-containing vacuole is an acidified lytic compartment. Aleurain is synthesized as a proenzyme. Proaleurain traffics through the Golgi to a comFig. I: Traffic of proteins to plant vacuoles. A schematic drawing of partment where it is processed to its mature form. Two ena portion of the endoplasmic reticulum (ER) and a Golgi complex is shown at the top; the arrows between ER and Golgi indicate that zymes are involved in this processing, and in vitro assays of vesicle traffic between the two organelles can procede in an antero- these enzymes demonstrated that they have optimal activity grade (to the right) or retrograde (to the left) direction. Soluble pro- around pH 5 (Holwerda et al., 1990). Purified aleurain also teins that enter the secretory pathway by translocation into the ER has a pH optimum of 5 (Holwerda and Rogers, 1992). When lumen during synthesis will, in general, move in anterograde vesicles expressed in tobacco suspension culture protoplasts and localto the Golgi complex and then transit through the Golgi complex to ized by subcellular fractionation, mature aleurain co-localizes be sorted into pathways with different destinations (Okita and Ro- with vacuolar enzyme markers (Holwerda et al., 1992). Figers, 1996). Soluble proteins lacking specific targeting determinants nally, as assessed by Western blot analysis, essentially all aleuwill enter vesicles that will fuse with the plasma membrane and release their contents to the cell exterior (indicated by arrow marked rain present in barley aleurone layers is in the mature form. Tonoplast intrinsic protein antibody markers: Subsequent «secretion»). In contrast, soluble proteins with vacuolar targeting determinants are sorted into pathways to vacuoles. Two separate studies of vacuole types were gready facilitated by the availvacuolar compartments, the lytic vacuole (LV) and protein storage ability of antibodies to two different tonoplast intrinsic provacuole (PSV) , and two separate vesicle pathways, clathrin coated teins (TIPs): to (X-TIP, present in tonoplast isolated from vesicles (CCVs) to the LV and smooth dense vesicles (SDVs) to the bean cotyledons Oohnson et al., 1989), and to tonoplast isoPSV; are shown. PV indicates a prevacuolar compartment for the LV lated from beetroot parenchyma vacuoles. The latter antisepathway. The numbers 1 and 2 indicate probable pathways for rum predominandy identified a tonoplast intrinsic protein of movement of membrane proteins to the vacuolar compartments: (1) the gamma class and was designated as anti-TIP-Ma27 (MarThe PSV may derive directly from specific regions of the endoty-Mazars et al., 1995). (X-TIP was thought to be seed speplasmic reticulum (ER) (Okita and Rogers, 1996; Robinson et aI., cific, but the original studies demonstrated its presence in 1995). (2) In some instances the LV and PSV compartments may merge (Paris et aI., 1996). a-TIP (indicated by «x» symbols on the root and shoot tissue for six days afrer germination, with PSV tonoplast) and TIP-Ma27 (indicated by star symbols on the LV gready decreased amounts in those tissues at eight days posttonoplast) are antibody markers for the two vacuole types. germination Oohnson et al., 1989). Our results with immunofluorescence studies on root tip cells (see below) suggest that the inability of Johnson et al. (1989) to detect (X-TIP in ally distinct vacuoles may exist separately within a plant cell, roots at later time points was due to a greater dilution of root and separate pathways carrying proteins to specific vacuolar tip cell protein by protein from elongated cells that lack compartments have been identified (Okita and Rogers, PSVs. 1996). In Figure 1 is presented a model to provide an oudine Two vacuole types during pea cotyledon development: Hoh et of current concepts about traffic of soluble proteins to various al. (1995) used anti-(X-TIP and anti-TlP-Ma27 antibodies to compartments in the plant cell secretory pathway. Biochemi- identify vacuole compartments in developing pea cotyledons. cal characterization of these pathways and of individual vacu- Immunogold EM studies demonstrated that protein body olar compartments may help in understanding how separate formation first occurred in tubular structures that were outtypes of vacuoles originate and are maintained by the cell. side of, and separate from, vegetative vacuoles. The structures containing protein bodies became labeled with anti-(X-TlP, while the vegetative vacuoles were labeled with anti-TlPFunctionally distinct protein storage and lytic vacuoles Ma27. Subcellular fractions from the developing cotyledons Aleurain-containing vacuoles distinct from protein storage were separated on isopycnic sucrose density gradients; memvacuoles: Studies to identify the intracellular localization of branes marked by (X-TIP banded at a density separate from

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Compartmentation of Proteins in Vacuoles

membranes marked by TIP-Ma27. Based on these observations, the authors postulated that PSVs and lytic (vegetative) vacuoles are separate organelles (Hoh et al., 1995). Distinct vacuolar compartments in root tip cells: We used the two different anti-TIP antibodies with immunofluorescence in confocal laser scanning microscopy to characterize vacuolar compartments in root tip cells from pea and barley (Paris et al., 1996). Anti-a-TIP antibodies labeled vacuolar structures that ranged in morphology from scattered spherical vacuoles to large cavernous and alveolar chambers. In contrast, antiTlP-Ma27 stained smaller spherical and tubular structures that had a worm-like or beads-on-a-string appearance. In double-labeling experiments with the two TIP antibodies, the two antigens were predominantly found on separate structures, although in some cells with larger vacuoles the two antigens appeared to be present on the same structures. We then used double-labeling experiments on barley root tip cells to determine the distribution of aleurain, a marker for an acidified lytic compartment, and barley lectin, a storage protein (Lerner and Raikhel, 1989) with respect to the two TIP antigens. Barley lectin was always found in vacuoles marked by anti-a-TlP; not all a-TIP vacuoles, however, contained barley lectin. This observation demonstrated that sub-populations of a-TIP vacuoles occur. In contrast, aleurain was always found in vacuoles marked by anti-TlP-Ma27. When barley lectin and aleurain were compared in double-label experiments, they were present in separate vacuoles. These results indicated that a-TIP was a marker for PSVs; although abundant in seeds, it was not seed-specific. TIPMa27 was a marker for an acidified, lytic compartment (Fig. 1). Clearly, individual cells could contain two separate vacuolar compartments and each compartment could be defined both by the TIP marker present in its tonoplast as well as by proteins present within the compartment. Presumably this arrangement would allow storage proteins to be segregated away from an acidic environment containing active proteases. If so, PSVs might have other characteristics that would contribute to stability of the storage proteins; one such characteristic could be a pH close to neutral. PSVs in aleurone cells have a near-neutral pH: The pH of PSVs was first determined in oat aleurone protoplasts; shortly afrer isolation the vacuolar pH was found to be 7, although it slowly declined over several days in culture as cells gradually accumulated larger vacuoles (Davies et al., 1996). Swanson and Jones similarly used a pH-sensitive fluorescent dye to measure PSV pH in barley aleurone protoplasts (Swanson and Jones, 1996). In protoplasts not treated with hormone, or protoplasts treated with ABA, the PSV pH was 6.6-6.8. However, when treated with gibberellin to activate synthesis of secreted hydrolytic enzymes, the PSV pH rapidly fell to the lower limit of measurement permitted by the dye, pH 5.8. Presumably the latter occurred because the cells had to mobilize amino acids for use in protein synthesis by providing an acidic environment where storage proteins could be degraded. All of these studies involved cells where protein storage occurred, and where a large central vacuole was not apparent. It is not clear if cells that contain the two separate vacuolar compartments differentiate into cells with a single central vacuole. However, our hypothesis is that a cell may merge the


two separate compartments to promote degradation of storage proteins, a process similar to what probably occurs in aleurone cells (Davies et al., 1996; Swanson and Jones, 1996), and then a central vacuole with diffent antigenic markers subsequently forms from the merged compartment.

Separate pathways to separate vacuolar compartments Soluble proteins sorted from the secretory pathway to the vacuole have two general types of vacuolar targeting signals (for reviews, see Nakamura and Matsuoka, 1993; Okita and Rogers, 1996). The first type, characterized on barley proaleurain and sweet potato prosporamin, contains a conserved central motif with the sequence Asn-Pro-Ile-Arg (NPIR), and mutation of the Ile to Gly abolishes vacuolar targeting (Nakamura et al., 1993). In contrast, targeting determinants within carboxy-terminal propeptides on barley lectin and tobacco chitinase have little or no sequence specificity because a high percentage of random amino acid sequences can provide targeting information (Dombrowski et al., 1993; Neuhaus et al., 1994). These facts would be most consistent with two different mechanisms for sorting proteins to the vacuole, one dependent upon recognition of a highly conserved amino acid sequence by a receptor and the other lacking sequence specificity but perhaps dependent upon physical characteristics of the proteins to be sorted, such as physical aggregation (Okita and Rogers, 1996). Matsuoka et al. (1995) studied the effects of wortmannin, an inhibitor of phosphatidylinositol kinases, on sorting of proteins carrying either the prosporamin NPIR targeting determinant or the barley lectin C-terminal propeptide targeting determinant in tobacco suspension culture cells. At concentrations that had little effect on vacuolar sorting of proteins carrying the NPIR targeting determinant, transport to the vacuole of proteins with the C-terminal propeptide was almost completely inhibited. These results provided a biochemical definition of two transport pathways from Golgi to the vacuole. Results from several laboratories indicated that two different vesicle populations were involved in transport of proteins from the Golgi to the vacuole (Fig. 1). Vesicles lacking a prominent cytoplasmic coat, with an osmophilic or dense appearance, have been observed apparently budding off from or in close association with the Golgi. These smooth, dense vesicles (SDVs) were shown by immunoEM to contain storage proteins (Hara-Nishimura et al., 1991; Herman, 1994; Kim et al., 1988; Robinson et al., 1995; zur Nieden et al., 1984). Clathrin coated vesicles (CCVs) are known to traffic between the Golgi and the vacuole in yeast. CCVs purified from developing pea cotyledons had been associated both with acid hydrolase enzymes (Harley and Beevers, 1989) as well as storage protein precursors (Harley and Beevers, 1989; Robinson et al., 1989). Recently it has become possible to purify SDVs and CCVs from pea cotyledons with little cross-contamination (Hohl et al., 1996; G. Hinz, pers. comm.). In these purified preparations, CCVs lack immunologically-detectable vicilin and legumin storage proteins that are abundant in SDVs.




A vacuolar sorting rectptor fonctions in the CCVpathway

We prepared two different types of antibodies to BP-SO: affinity-purified polyclonal rabbit antibodies raised to a synThe knowledge that the NPIR motif was highly conserved thetic peptide representing the 20 amino acids at the N-terin proaleurain and prosporamin suggested the possibility that minus of the protein, and a mouse monoclonal antibody to it is recognized by a vacuolar sorting receptor. Accordingly, an unknown epitope elsewhere on the protein. When used in we constructed an affinity column with a synthetic peptide immunofluorescence experiments with pea root tip cells, the carrying the vacuolar targeting sequence from proaleurain antibodies co-localized on punctate and small spherical struc(Holwerda et al., 1992), and tested detergent lysates of pea tures that were diffusely distributed throughout the cell cyCCV membranes for a protein that would recognize that toplasm. Importandy, the tonoplast of PSVs and lytic vacupeptide but not a control peptide. A single SO kDa protein oles was not labeled by either antibody. ImmunoEM studies bound specifically and could be eluted at pH 4; this was with the antipeptide antibodies demonstrated specific labeling over dilated ends of Golgi cisternae and over what we identified as BP-SO (Kirsch et al., 1994). In vitro binding studies of purified BP-SO demonstrated a termed «prevacuoles»; the latter were -250 nm diameter vacK.:t of 37 nM for the proaleurain vacuolar targeting determi- uoles adjacent to much larger vacuoles that in some cases apnant peptide. A decapeptide carrying the prosporamin target- peared to be in the process of fusing with the larger vacuoles ing determinants competed weakly with the proaleurain pep- (Paris et al., 1997). tide for binding, but a mutated form in which NPIR was Within the cytoplasmic domains of the VSR proteins is changed to NPGR did not compete for binding. A synthetic the sequence YMPL that conforms to the Tyr-X-X-0 (where peptide representing the barley lectin propeptide sequence X is any amino acid and 0 is hydrophobic with a bulky side also did not compete for binding. The pH optimum for chain) motif implicated in binding adaptor proteins as part of binding was 6-6.5, and binding was abolished at pH 4. Sim- the process of assembling CCVs (Ohno et al., 1995). In genilar results to test binding specificity were subsequendy ob- eral, the cytoplasmic domains of the proteins are highly contained using different synthetic peptides on affinity columns served within the first ~ of their length and then diverge; the (Kirsch et al., 1996). Tyr-X-X-0 motif is within the conserved region (Paris et al., When intact vesicles carrying BP-SO were treated with pro- 1997) indicating that all are likely to be incorporated into teinase K, the molecular mass of the protein decreased by CCVs. Supporting this prediction, highly purified prepara-5 kDa. When the treated vesicles were lysed, the shortened tions of pea CCVs that lack immunologically-detectable storprotein bound to the affinity column, and its amino-terminal age proteins are highly enriched with BP-SO antigen, while sequence was the same as intact BP-SO. These results indi- highly purified preparations of pea SDVs enriched in vicilin cated that BP-SO had a short cytoplasmic tail accessible to the and legumin lack immunologically-detectable BP-SO (G. protease, but that the N-terminal portion within the vesicle Hinz, pers. comm.). lumen contained the ligand binding domain (Kirsch et al., Thus, the following points argue strongly that BP-SO is a 1994). functional vacuolar sorting receptor for proteins carrying the Further information has come with the cloning of cDNAs NPIR type of vacuolar targeting determinant: (1) BP-SO for BP-SO and homologous proteins (Paris et al., 1997). BP- binds peptide ligands that represent functionally defined vacSO is a member of what we have termed the «VSR multigene uolar targeting determinants, and mutation of functionally f.unily», of which at least four are expressed in developing pea essential residues abolishes binding. (2) The pH requirements seeds and three are expressed in Arabidopsis. These gene se- for binding, maximal at pH 6-6.5 and abolished at pH 4 are quences are also highly conserved in maize and rice. The VSR those expected for a protein binding ligand in the Golgi and proteins in general are 623 amino acids in length, including a releasing it in an acidified prevacuolar compartment. (3) BPsignal peptide; a single transmembrane domain is present SO is present in Golgi and prevacuoles. (4) BP-SO is present with a carboxy-terminal cytoplasmic tail of -37 amino acids. in CCVs and not SDVs. We suggest that the multiple members of the VSR family They all contain three Epidermal Growth Factor (EGF) repeats in the lumenal portion near the transmembrane do- function together to provide a mechanism for sorting a broad main; one of these repeats is predicted to bind two calcium population of proteins to the lytic vacuole. Thus, BP-SO and molecules and may contain a hydroxylated Asn residue (Paris closely related homologues may prefer to bind proteins with et al., 1997). The first -410 amino acids of the VSR proteins the central NPIR motif, but other homologues may have a comprise a unique sequence without database homologues different ligand binding specificity. As systems for expressing outside of the VSR family; specifically there is no homology and purifying truncated forms of the various homologues to the VPS lOp yeast vacuolar sorting receptor (Marcusson et that lack transmembrane and cytoplasmic domains are develal., 1994). Some divergence in the sequences is apparent oped, it will be possible to develop binding assays that will alwhen alignment comparisons are made after removal of the low this hypothesis to be tested. It will be of particular intersignal peptides. For example, the sequence of Arabidopsis est to learn how storage proteins destined for SDVs and the Z3S123 is 72 % identical to that of BP-SO, while Arabidopsis PSV are segregated away and escape binding by the VSR MJ447 is S2 % identical (Paris et al., 1997). A truncated form family of receptors. of BP-SO, lacking transmembrane and cytoplasmic domains, was secreted from the cells when expressed in tobacco suspenAcknowledgements sion culture protoplasts (Paris et al., 1997). This result conThis research was supported by grants DE-FG9SER20165 from firms the predictions of a single transmembrane domain the Department of Energy and GM52427 from the National Inmade from sequence analyses. stitutes of Health.

Compartmentation of Proteins in Vacuoles

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