Mass transport of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplasmic reticulum-derived vesicle is involved in protein mobilization in germinating seeds

A vacuolar cysteine proteinase, designated SH-EP, is expressed in the cotyledon of germinated Vigna mungo seeds and is responsible for the degradation of storage proteins. SH-EP is a characteristic vacuolar proteinase possessing a COOH-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In this work, immunocytochemical analysis of the cotyledon cells of germinated V. mungo seeds was performed using seven kinds of antibodies to identify the intracellular transport pathway of SH-EP from ER to protein storage vacuoles. A proform of SH-EP synthesized in ER accumulated at the edge or middle region of ER where the transport vesicle was formed. The vesicle containing a large amount of proSH-EP, termed KV, budded off from ER, bypassed the Golgi complex, and was sorted to protein storage vacuoles. This massive transport of SH-EP via KV was thought to mediate dynamic protein mobilization in the cotyledon cells of germinated seeds. We discuss the possibilities that the KDEL sequence of KDEL-tailed vacuolar cysteine proteinases function as an accumulation signal at ER, and that the mass transport of the proteinases by ER-derived KV-like vesicle is involved in the protein mobilization of plants.     

C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase

Sulfhydryl-endopeptidase (SH-EP) is a papain-type vacuolar proteinase expressed in cotyledons of germinated Vigna mungo seeds, and the enzyme possesses a C-terminal propeptide containing KDEL tail, an endoplasmic reticulum retention signal for soluble proteins. SH-EP is transported to vacuoles via a KDEL vesicle (KV) through a Golgi complex-independent route. To see the function of the KDEL sequence of SH-EP, wild-type SH-EP and its KDEL deletion mutant (SH-EPDeltaKDEL) were heterologously expressed in Arabidopsis and in cultured tobacco Bright Yellow 2 cells, and their intracellular transport pathways and localizations were analyzed. A combination of the results from analyses for transformed Arabidopsis and tobacco (Nicotiana tabacum) cells indicated that wild-type SH-EP is packed into KV-like vesicles through the KDEL sequence and is transported to vacuoles in the cells of transformants. In contrast, KV was not formed/induced in the cells expressing SH-EPDeltaKDEL, and the mutant protein was mainly secreted. Therefore, the C-terminal KDEL sequence of the KDEL-tailed cysteine proteinase is thought to be involved in the formation of KV, and in the efficient vacuolar transport of the proteins through KV.     

Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP).

A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages.     

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.     

Involvement of gibberellins in expression of a cysteine proteinase (SH-EP) in cotyledons of Vigna mungo seedlings.

The expression of a papain-type proteinase, designated SH-EP, in cotyledons of Vigna mungo seedlings has been shown to require some factors in the embryonic axes. Gibberellin A1 (GA(1)) and GA(20) were identified by GC-MS in embryonic axes of V. mungo seedlings. The level of accumulation of SH-EP in cotyledons of V. mungo seedlings was greatly reduced by treatment of the seeds with uniconazole-P, an inhibitor for GA biosynthesis. The reduced level of accumulation of SH-EP in cotyledons by uniconazole-P was recovered by exogenous application of GA(1) and GA(20) to the seedlings.     

Posttranslational removal of the carboxyl-terminal KDEL of the cysteine protease SH-EP occurs prior to maturation of the enzyme

SH-EP is a cysteine protease from germinating mung bean (Vigna mungo) that possesses a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In order to examine the function of the ER retention sequence, we expressed a full-length cDNA of SH-EP and a minus-KDEL control in insect Sf-9 cells using the baculovirus system. Our observations on the synthesis, processing, and trafficking of SH-EP in Sf-9 cells suggest that the KDEL ER-retention sequence is posttranslationally removed either while the protein is still in the ER or immediately after its exit from the ER, resulting in the accumulation of proSH-EP minus its KDEL signal. It is this intermediate form that appears to progress through the endomembrane system and is subsequently processed to form mature active SH-EP. The removal of an ER retention may regulate protein delivery to a functional site and present an alternative role for ER retention sequences in addition to their well established role in maintaining the protein composition of the ER lumen.     

Molecular cloning and characterization of Vigna mungo processing enzyme 1 (VmPE-1), an asparaginyl endopeptidase possibly involved in post-translational processing of a vacuolar cysteine endopeptidase (SH-EP)

Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxy side of asparagine residues. Vigna mungo processing enzyme 1, termed VmPE-1, occurs in the cotyledons of germinated seeds of V. mungo, and is possibly involved in the post-translational processing of a vacuolar cysteine endopeptidase, designated SH-EP, which degrades seed storage protein. VmPE-1 also showed a substrate specificity to asparagine residues, and its enzymatic activity was inhibited by NEM but not E-64. In addition, purified VmPE-1 had a potential to process the recombinant SH-EP precursor to its intermediate in vitro. cDNA clones for VmPE-1 and its homologue, named VmPE-1A, were identified and sequenced, and their expressions in the cotyledons of V. mungo seedlings and other organs were investigated. VmPE-1 mRNA and SH-EP mRNA were expressed in germinated seeds at the same stage of germination although the enzymatic activity of VmPE-1 rose prior to that of SH-EP. The level of VmPE-1A mRNA continued increasing as germination proceeded. In roots, stems and leaves of fully grown plants, and in hypocotyls, VmPE-1 and VmPE-1A were little expressed. We discuss possible functions of VmPE-1 and VmPE-1A in the cotyledons of germinated seeds.     

The cargo in vacuolar storage protein transport vesicles is stratified

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.     

Lack of a vacuolar sorting receptor leads to non-specific missorting of soluble vacuolar proteins in Arabidopsis seeds

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.     

Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds

Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to MP23 includes six putative membrane-spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. These two TIPs might have a specific function during the maturation of pumpkin seeds.     

Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.     

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns,intracellular localization and functions in globulin proteolysis

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V. sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (VPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.     

Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.     

A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.     

Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor

BP-80, later renamed VSR(PS-1), is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR(PS-1) characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR(PS-1) together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR(PS-1) is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR(PS-1) therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR(PS-1) receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent.     

A vacuolar sorting receptor PV72 on the membrane of vesicles that accumulate precursors of seed storage proteins (PAC vesicles)

A novel vesicle, referred to as a precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors to protein storage vacuoles in maturing pumpkin seeds. PV72, a type I integral membrane protein with three repeats of epidermal growth factor, was found on the membrane of the PAC vesicles. PV72 had an ability to bind to pro2S albumin, a storage protein precursor, in a Ca(2+)-dependent manner, via the C-terminal region of pro2S albumin, which was found to function as a vacuolar targeting signal. This implies that PV72 is a vacuolar sorting receptor of the storage protein. PV72 was specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins. Subcellular fractionation showed that PV72 was also accumulated in the microsomal fraction. A fusion protein consisting of GFP and the transmembrane domain and the cytosolic tail of PV72 was localized in Golgi complex. PV72 in the isolated PAC vesicles had a complex type of oligosaccharide, indicating that PV72 passed though the Golgi complex. These results suggest that PV72 is recycled between PAC vesicles and Golgi complex/post-Golgi compartments. PV72 appears to be responsible for recruiting pro2S albumin molecules from the Golgi complex to the PAC vesicles.     

Development of Endopeptidase Activity in Cotyledons of Vigna mungo Seedlings: Effects of Exogenously Applied End-Products and Plant Hormones

The development of endopeptidase activity in cotyledons of Vignamungo seedlings was examined after application of exogenousamino acids, sugars and plant hormones. The endopeptidase activityin the cotyledons fell when germinating seeds were allowed toabsorb a solution of amino acids at high concentrations, andit was postulated that this effect might have been caused inpart by osmotic stress and in part by end-product repression.Protein immunoblotting with an antiserum against SH-EP, themajor cysteine endopeptidase occurring in the cotyledons, showedthat sugars and amino acids at high concentrations also delayedthe post-translational processing of SH-EP intermediates. Endopeptidaseactivity equivalent to nearly twice that in controls was observedwhen GA₃ was applied at 10 to 100 µM to cotyledons thathad been detached from the embryonic axis. In addition, naphthaleneaceticacid at 1 to 100 µM, kinetin at 1 to 10 µM and jasmonicacid at 1 to 10 µM also increased the activity to a limitedextent. Results of pulse-chase experiments suggested that theeffect of GA₁ on the endopeptidase activity in the detachedcotyledons was attributable to suppression of the degradationof the enzyme. Protein immunoblotting revealed the presenceof 34-kOa and 35-kDa intermediates of SH-EP in addition to previouslyreported 36-kDa and 43-kDa intermediates. (Received June 26, 1995; Accepted October 16, 1995)     

An ER-localized form of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves

Putative vacuolar sorting receptors that bind to the vacuolar targeting signals have been found in various plants; pumpkin PV72, pea BP-80 and Arabidopsis AtELP. PV72 is a seed-specific receptor that is predicted to sort seed storage proteins to protein storage vacuoles. Analysis by surface plasmon resonance showed that the lumenal domain of PV72 bound to an NPIR (a typical vacuolar targeting signal)-containing peptide of the precursor of a cysteine proteinase, AtALEU, in the presence of Ca(2+) (K(D) = 0.1 micro M). To elucidate the receptor-dependent transport of vacuolar proteins in plant cells, we produced transgenic Arabidopsis plants that expressed a fusion protein (PV72-HDEL) composed of the lumenal domain of PV72 and an endoplasmic reticulum (ER)-retention signal, HDEL. The expression of PV72-HDEL induced the accumulation of the AtALEU precursor. The accumulation level of the AtALEU precursor was dependent on that of PV72-HDEL. In contrast, it did not induce the accumulation of a precursor of another cysteine proteinase, RD21, which contains no NPIR. Detailed subcellular localization revealed that both the AtALEU precursor and PV72-HDEL accumulated in the ER fraction. We found that most of the AtALEU precursor molecules formed a complex with PV72-HDEL. The AtALEU precursor might be trapped by PV72-HDEL in the ER and not transported to the vacuoles. This in-planta analysis supports the hypothesis that an Arabidopsis homolog of PV72 functions as a sorting receptor for the NPIR-containing proteinase. The overall results suggest that vacuolar sorting receptors for the protein storage vacuoles and the lytic vacuoles share the similar recognition mechanism for a vacuolar targeting signal.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.