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.     

AtVPS29, a putative component of a retromer complex, is required for the efficient sorting of seed storage proteins

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) as larger precursors and are sorted to protein storage vacuoles, where they are converted into the mature forms. We report here an Arabidopsis mutant, maigo 1 (mag1), which abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag1 seeds mis-sort storage proteins by secreting them from cells. mag1 seeds have smaller protein storage vacuoles in the seeds than do wild-type seeds. The MAG1 gene encodes a homolog of the yeast (Saccharomyces cerevisiae) protein VPS29. VPS29 is a component of a retromer complex for recycling a vacuolar sorting receptor VPS10 from the pre-vacuolar compartment to the Golgi complex. Our findings suggest that MAG1/AtVPS29 protein is involved in recycling a plant receptor for the efficient sorting of seed storage proteins. The mag1 mutant exhibits a dwarf phenotype. A plant retromer complex plays a significant role in plant growth and development.     

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.     

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.     

Two short sequences from amaranth 11S globulin are sufficient to target green fluorescent protein and beta-glucuronidase to vacuoles in Arabidopsis cells.

Vacuolar sorting of seed storage proteins is a very complex process since several sorting pathways and interactions among proteins of different classes have been reported. In addition, although the C-terminus of several 7S proteins is important for vacuolar delivery, other signals seem also to be involved in this process. In this work, the ability of two sequences of the Amaranthus hypochondriacus 11S globulin (amaranthin) to target reporter proteins to vacuoles was studied. We show that the C-terminal pentapeptide (KISIA) and the GNIFRGF internal sequence fused at the C terminal region of genes encoding secretory versions of green fluorescent protein (GFP) and GFP-beta-glucuronidase (GFP-GUS) were sufficient to redirect these reporter proteins to the vacuole of Arabidopsis cells. According to the three-dimensional structure of 7S and 11S storage globulins, this internal vacuolar sorting sequence corresponds to the alpha helical region involved in trimer formation, and is conserved within these families. In addition, these sequences were able to interact in vitro, in a calcium dependent manner, with the sunflower vacuolar sorting receptor homolog to pea BP-80/AtVSR1/pumpkin PV72. This work shows for the first time the role of a short internal sequence conserved among 7S and 11S proteins in vacuolar sorting.     

Functional Identification of Sorting Receptors Involved in Trafficking of Soluble Lytic Vacuolar Proteins in Vegetative Cells of Arabidopsis

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis βFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.Two different types of vacuoles have been identified in plant cells. One of them is the lytic vacuole (LV) that is present in vegetative cells, and the other is the protein storage vacuole (PSV) that is present in seed cells (Frigerio et al., 2008; Zouhar and Rojo, 2009; De Marcos Lousa et al., 2012). These two types of vacuoles have different functions. The LV carries out various functions such as osmotic pressure regulation, various hydrolytic activities, detoxification, and homeostasis of calcium and sodium ions. For some of these aspects LV is analogous to the vacuole in yeast (Saccharomyces cerevisiae) or lysosomes in animal cells. In contrast, the PSV is unique in plants and stores a large amount of proteins and minerals that are necessary for seed germination. To perform these functions, vacuoles need a large number of proteins.The organellar proteins destined for vacuoles have to be transported from the endoplasmic reticulum (ER) via a process called protein trafficking. This has been extensively studied in many different eukaryotic cell types, including plant cells. In general, proteins that belong to various endomembrane compartments are cotranslationally translocated into the ER and then transported through the Golgi apparatus and other intermediate compartments depending on their final destinations (Jurgens, 2004; Jolliffe et al., 2005; Sato and Nakano, 2007; Hwang and Robinson, 2009; Reyes et al., 2011). Vesicles are used to transport proteins from one compartment to another. Another important aspect is the specific targeting of organellar proteins. For this, organellar proteins carry a specific sorting or targeting signal that can be a sequence motif generated intrinsically or added posttranslationally (Hadlington and Denecke, 2000; Robinson et al., 2005; Hwang, 2008). The sequence motifs are recognized specifically by sorting receptors localized at the organelles that serve as donor compartments in trafficking pathways (Bassham and Raikhel, 2000; De Marcos Lousa et al., 2012).Two different types of sorting receptors, receptor homology-transmembrane-RING H2 domain proteins (RMRs) and vacuolar sorting receptors (VSRs), have been shown to be involved in the trafficking of vacuolar proteins. It has been proposed that RMRs function as a sorting receptor for storage proteins (Park et al., 2005; Hinz et al., 2007; Wang et al., 2011a). RMRs are type I membrane proteins and those in the luminal domain specifically interact with the C-terminal vacuolar sorting sequence (ctVSS) of storage proteins (Park et al., 2005; Shen et al., 2011). In addition, overexpression of an AtRMR1 deletion mutant inhibits the trafficking of phaseolin to the PSV, but not the protein trafficking to the LV, in protoplasts from leaf cells (Park et al., 2005). VSRs have been identified from various plant species and shown to specifically interact with the sorting motif of vacuolar proteins, which is known as the sequence-specific vacuolar sorting signal (ssVSS) or N-terminal propeptide (Ahmed et al., 1997; Hadlington and Denecke, 2000; Masclaux et al., 2005; Robinson et al., 2005; Hwang, 2008). In plant cells, the majority of VSRs localize to the prevacuolar compartment (PVC), which is the intermediate organelle between the trans-Golgi network (TGN) and vacuole (Tse et al., 2004; daSilva et al., 2005; Miao et al., 2006). In addition, a minor portion of VSR1 localizes to the TGN in plant cells, which supports the notion that VSRs recycle to the TGN from the PVC for sorting of their cargo proteins (Kim et al., 2010). Recent studies in plant cells questioned this concept and proposed other mechanisms for sorting vacuolar proteins. In the alternative proposal, sorting of vacuolar proteins may occur at the ER, and the VSRs may recycle from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). VSRs that were once thought to function as sorting receptors at the TGN for the LV proteins (daSilva et al., 2005; Foresti et al., 2010; Kim et al., 2010) have an additional function in the protein trafficking to the PSV in seed cells (Shimada et al., 2003; Zouhar et al., 2010). By using a genetic approach, it has been shown that among seven Arabidopsis (Arabidopsis thaliana) VSRs, VSR1, VSR3, and VSR4 play a role in trafficking of 12S globulins and 2S albumins in seed cells.The VSR isoforms involved in the protein trafficking to the PSV also exist in vegetative tissues (Laval et al., 1999; Kim et al., 2010; Zouhar et al., 2010). Mutations in both VSR1 and VSR4 cause secretion of AtAleurain, but not other LV proteins, into the apoplasts. Thus, it is not clearly understood what is the physiological role of AtVSRs in vegetative tissues (except for their role in vacuolar trafficking of AtAleurain), and what are the VSRs of other vacuolar proteins. In previous studies, it was demonstrated that overexpression of mutant forms of VSR1, VSR2, or BP80 of pea (Pisum sativum), a close homolog of VSR3 and VSR4, in protoplasts from wild-type plants affects trafficking of proteins to the LV (daSilva et al., 2005; Foresti et al., 2010; Kim et al., 2010). In this study, we utilized various VSR and RMR mutant plants and examined the effect of these mutations on the trafficking of LV and PSV proteins in protoplasts. These studies demonstrated that VSR1, VSR3, and VSR4, but not other VSRs and RMRs, are involved in trafficking of soluble LV and PSV proteins in vegetative cells. Further, the luminal domain but not the cytosolic tail of VSRs contains the determinant for the sorting specificity.     

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K⁺ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K⁺ homeostasis.     

Functional specialization within the vacuolar sorting receptor family: VSR1, VSR3 and VSR4 sort vacuolar storage cargo in seeds and vegetative tissues

Two different gene families have been proposed to act as sorting receptors for vacuolar storage cargo in plants: the vacuolar sorting receptors (VSRs) and the receptor homology‐transmembrane‐RING H2 domain proteins (RMRs). However, functional data on these genes is scarce and the identity of the sorting receptor for storage proteins remains controversial. Through a genetic screen we have identified the mtv2 mutant, which is defective in vacuolar transport of the storage cargo VAC2 in shoot apices. Map‐based cloning revealed that mtv2 is a loss of function allele of the VSR4 gene. We show that VSR1, VSR3 and VSR4, but not the remaining VSRs or RMRs, participate in vacuolar sorting of VAC2 in vegetative tissues, and 12S globulins and 2S albumins in seeds, an activity that is essential for seedling germination vigor. Finally, we demonstrate that the functional diversification in the VSR family results from divergent expression patterns and also from distinct sorting activities of the family members.     

Rha1, an Arabidopsis Rab5 homolog,plays a critical role in the vacuolar trafficking of soluble cargo proteins

Rab proteins are members of the Ras superfamily of small GTP binding proteins and play important roles in various intracellular trafficking steps. We investigated the role of Rha1, an Arabidopsis Rab5 homolog, in intracellular trafficking in Arabidopsis protoplasts. In the presence of a dominant-negative mutant of Rha1, soluble vacuolar cargo proteins such as sporamin:green fluorescent protein (Spo:GFP) and Arabidopsis aleurain like protein:GFP are not delivered to the central vacuole; instead, they accumulate as a diffuse or punctate staining pattern within the cell. Spo:GFP at the punctate stains observed in the presence of hemagglutinin:Rha1[S24N] is colocalized with endogenous vacuolar sorting receptor (VSR(At-1)), which is known to localize primarily to the prevacuolar compartment, whereas Spo:GFP in the diffuse pattern is associated with tonoplasts. Furthermore, expression of Rha1[S24N] causes the secretion of a portion of the vacuolar proteins into medium. However, the inhibitory effect of Rha1[S24N] on vacuolar trafficking is relieved partially by coexpressed wild-type Rha1. Based on these results, we propose that Rha1 plays a critical role in the trafficking of soluble cargoes from the prevacuolar compartment to the central vacuole.     

An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1–AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.Membrane trafficking in plants shares many fundamental features with those in yeast and animals (Bassham et al., 2008). In general, vacuolar proteins are synthesized on the rough endoplasmic reticulum and then transported to vacuoles via the Golgi apparatus (Xiang et al., 2013; Robinson and Pimpl, 2014). The vacuolar trafficking in plants has been studied by monitoring the transport of reporter proteins to lytic vacuoles in vegetative cells and tissues (Jin et al., 2001; Pimpl et al., 2003; Miao et al., 2008; Niemes et al., 2010). Recently, seed storage proteins became a model cargo for monitoring the transport of endogenous vacuolar proteins in plants (Shimada et al., 2003a; Sanmartín et al., 2007; Isono et al., 2010; Pourcher et al., 2010; Uemura et al., 2012; Shirakawa et al., 2014). During seed maturation, a large amount of storage proteins are synthesized and sorted to specialized vacuoles, the protein storage vacuoles (PSVs). To properly deliver vacuolar proteins, sorting receptors play a critical role in recognizing the vacuole-targeting signal of the proteins. VACUOLAR PROTEIN SORTING10 and Man-6-P receptor function as sorting receptors for vacuolar/lysosomal proteins in the trans-Golgi network (TGN) of yeast and mammals, respectively. The best-characterized sorting receptors in plants are VACUOLAR SORTING RECEPTOR (VSR) family proteins (De Marcos Lousa et al., 2012). VSRs have been shown to function in sorting both storage proteins to PSVs (Shimada et al., 2003a; Fuji et al., 2007) and lytic cargos to lytic vacuoles (Zouhar et al., 2010).To sort the receptors in the TGN into vacuoles/lysosomes, the adaptor protein (AP) complex binds the cytosolic domain of the receptors. The AP complexes form evolutionarily conserved machinery that mediates the post-Golgi trafficking in eukaryotic cells (Robinson, 2004). There are five types of AP complexes, AP-1 to AP-5. The functions of AP-1, AP-2, and AP-3 have been established. AP-1 appears to be involved in trafficking between the TGN and endosomes (Hirst et al., 2012), AP-2 is involved in clathrin-mediated endocytosis (McMahon and Boucrot, 2011), and AP-3 is involved in protein trafficking from the TGN/endosomes to the vacuole/lysosomes (Dell’Angelica, 2009). However, little is known about AP-4 and AP-5. Mammalian AP-4 may be involved in basolateral sorting in polarized cells and in the transport of specific cargo proteins, such as the amyloid precursor protein APP, from the TGN to endosomes (Burgos et al., 2010). The fifth AP complex, AP-5, was recently identified, and its orthologs are widely conserved in the eukaryotic genomes (Hirst et al., 2011). The AP complexes exist as heterotetrameric proteins that consist of two large subunits (β1-5 and one each of ɣ/α/δ/ε/ζ), one medium subunit (µ1-5), and one small subunit (σ1-5). The sorting mechanism is best characterized for the medium (µ) subunit, which is known to recognize the Tyr-based YXXФ motif (where Ф represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al., 1995). Mutations of the YXXФ motif abolish the interaction with µ and alter the subcellular localization of the cargo proteins.The genome of Arabidopsis (Arabidopsis thaliana) contains all five sets of putative AP genes (Bassham et al., 2008; Hirst et al., 2011). The function of AP-4 in membrane trafficking and its physiological roles in plants are largely unknown. In this study, we identified and characterized the AP-4 complex in Arabidopsis. Mutants lacking the AP-4 subunits exhibited defects in VSR1-mediated vacuolar sorting of storage proteins in seeds. Our results provide new insights into the receptor-mediated vacuolar trafficking in post-Golgi pathways.     

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.     

Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component.

More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface. A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins. Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole. Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature. Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates. Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained. A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP. Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.     

Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells

We generated fusions between three Arabidopsis (Arabidopsis thaliana) tonoplast intrinsic proteins (TIPs; alpha-, gamma-, and delta-TIP) and yellow fluorescent protein (YFP). We also produced soluble reporters consisting of the monomeric red fluorescent protein (RFP) and either the C-terminal vacuolar sorting signal of phaseolin or the sequence-specific sorting signal of proricin. In transgenic Arabidopsis leaves, mature roots, and root tips, all TIP fusions localized to the tonoplast of the central vacuole and both of the lumenal RFP reporters were found within TIP-delimited vacuoles. In embryos from developing, mature, and germinating seeds, all three TIPs localized to the tonoplast of protein storage vacuoles. To determine the temporal TIP expression patterns and to rule out mistargeting due to overexpression, we generated plants expressing YFP fused to the complete genomic sequences of the three TIP isoforms. In transgenic Arabidopsis, gamma-TIP expression was limited to vegetative tissues, but specifically excluded from root tips, whereas alpha-TIP was exclusively expressed during seed maturation. delta-TIP was expressed in vegetative tissues, but not root tips, at a later stage than gamma-TIP. Our findings indicate that, in the Arabidopsis tissues analyzed, two different vacuolar sorting signals target soluble proteins to a single vacuolar location. Moreover, TIP isoform distribution is tissue and development specific, rather than organelle specific.     

Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae

Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.     

Isolation and characterization of Aspergillus oryzae vacuolar protein sorting mutants

The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenk?rper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenk?rper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.     

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.     

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.