FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.The plant vacuole is the largest organelle in a plant cell in which proteins, metabolites, and ions can be stored or sequestered. The vacuole is essential for plant development and growth and is directly or indirectly involved in various biotic and abiotic stress responses (Zhang et al., 2014). The vacuole is also the central organelle for degradation of endocytic and autophagic protein substrates through the activity of vacuolar proteases. In both degradation pathways, substrates are transported to the vacuole by intracellular membrane trafficking. In endocytic degradation, plasma membrane-localized proteins are targeted to the vacuole for degradation by endosomes (Reyes et al., 2011). This process is important, among others, to control the abundance of plasma membrane receptors and thus downstream signaling events. Autophagic degradation is mainly involved in nutrient recycling. During this process, cytosolic proteins and organelles are either selectively or nonselectively transported by double membrane autophagosomes to the vacuole to be degraded (Liu and Bassham, 2012). Vacuolar transport defines an intracellular transport pathway by which de novo synthesized proteins or metabolic compounds are carried to the vacuole by vesicle transport (Drakakaki and Dandekar, 2013).In yeast (Saccharomyces cerevisiae), forward genetic screens aimed at finding mutants with defective vacuolar transport or vacuolar morphology have identified more than 30 VACUOLAR PROTEIN SORTING (VPS) and VACUOLAR MORPHOLOGY (VAM) genes (Banta et al., 1988; Raymond et al., 1992; Wada and Anraku, 1992). Closer analyses have shown that many of these mutants have defects both in protein sorting and in vacuole biogenesis, suggesting a close link between these processes. vps and vam mutants were classified into six mutant classes according to their phenotypes. The strategic success of these screens has been confirmed when later studies revealed that many of the genes categorized in the same mutant class were coding for subunits of the same protein complexes. Among them were complexes important for membrane transport and fusion events, such as the endosomal sorting complex required for transport (ESCRT)-I to ESCRT-III (Henne et al., 2011) or the homotypic fusion and vacuole protein sorting (HOPS) complex (Balderhaar and Ungermann, 2013).Sequence homologs of most yeast VPS genes can be found in the Arabidopsis (Arabidopsis thaliana) genome (Sanderfoot and Raikhel, 2003; Bassham et al., 2008), and some of them were reported to be involved in intracellular trafficking as well as vacuole biogenesis. For example, the Arabidopsis vacuoleless (vcl)/vps16 mutant is embryo lethal and lacks lytic vacuoles (Rojo et al., 2001). VPS16 is a subunit of the HOPS complex, suggesting that membrane fusion events mediated by VCL/VPS16 are also important for plant vacuole biogenesis. Several other Arabidopsis vps mutants were also shown to have altered vacuole morphology at the mature embryo stage (Shimada et al., 2006; Sanmartín et al., 2007; Ebine et al., 2008, 2014; Yamazaki et al., 2008; Zouhar et al., 2009; Shahriari et al., 2010), showing that there is a conserved mechanism regulating vacuolar transport and vacuole biogenesis. However, in contrast to yeast, in which mutants without vacuole or severe biogenesis defects are viable, plant vacuoles seem to be essential for plant development.We have previously shown that defects in the deubiquitinating enzyme (DUB) ASSOCIATED MOLECULE WITH THE Src homology-3 DOMAIN OF STAM3 (AMSH3) also lead to a severe vacuole biogenesis defect (Isono et al., 2010). AMSH homologs do not exist in budding yeast but are conserved in animals and plants. Our previous studies have shown that AMSH3 can directly interact with ESCRT-III subunits (Katsiarimpa et al., 2013). ESCRT-III is a multiprotein complex that is essential for multivesicular body (MVB) sorting (Winter and Hauser, 2006) and hence for plant growth and development (Haas et al., 2007; Spitzer et al., 2009; Katsiarimpa et al., 2011; Cai et al., 2014). AMSH proteins regulate intracellular trafficking events, including endocytic degradation, vacuolar transport, and autophagic degradation through its interaction with ESCRT-III (Isono et al., 2010; Katsiarimpa et al., 2011, 2013, 2014). Prior to our characterization of the amsh3 mutant, AMSH proteins had not been implicated in vacuole biogenesis. Thus, we reasoned that there might be additional, yet unidentified, factors important for regulating vacuole biogenesis in plants. Further, we reasoned that other mutants with a defect in vacuole biogenesis, analogous to amsh3, might also exhibit seedling lethality.Thus, with the goal to identify and characterize these factors, we carried out a two-step mutant screen. We first selected seedling lethal mutants from an ethyl methansulfonate (EMS)-mutagenized population and then examined the vacuole morphology in these mutants. The isolated mutants were designated vacuolar fusion defective (vfd). vfd1 is affected in the expression of a functional Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing FYVE1 protein. FYVE1 was originally identified in silico as one of 16 FYVE domain-containing proteins in Arabidopsis with no apparent homologs in yeast and mammals (van Leeuwen et al., 2004). FYVE domains bind phosphatidylinositol 3-P, a phospholipid that is a major constituent of endosomal membranes. Hence, FYVE domain-containing proteins are implicated in intracellular trafficking (van Leeuwen et al., 2004; Wywial and Singh, 2010). In a previous work, we have shown that a null mutant of FYVE1, fyve1-1, is defective in IRON-REGULATED TRANSPORTER1 (IRT1) polarization and that FYVE1 is essential for plant growth and development (Barberon et al., 2014). A very recent publication describing the same mutant has shown that FYVE1/FYVE domain protein required for endosomal sorting1 (FREE1) is also important for the early and late endosomal trafficking events (Gao et al., 2014). In this study, we show that FYVE1 is also regulating ubiquitin-dependent membrane protein degradation, vacuolar transport, autophagy, and vacuole biogenesis. Altogether, our results point toward FYVE1 being a key component of the intracellular trafficking machinery in plants.     

Homomeric Interaction of AtVSR1 Is Essential for Its Function as a Vacuolar Sorting Receptor

Vacuolar sorting receptors, BP80/VSRs, play a critical role in vacuolar trafficking of soluble proteins in plant cells. However, the mechanism of action of BP80 is not well understood. Here, we investigate the action mechanism of AtVSR1, a member of BP80 proteins in Arabidopsis (Arabidopsis thaliana), in vacuolar trafficking. AtVSR1 exists as multiple forms, including a high molecular mass homomeric complex in vivo. Both the transmembrane and carboxyl-terminal cytoplasmic domains of AtVSR1 are necessary for the homomeric interaction. The carboxyl-terminal cytoplasmic domain contains specific sequence information, whereas the transmembrane domain has a structural role in the homomeric interaction. In protoplasts, an AtVSR1 mutant, C2A, that contained alanine substitution of the region involved in the homomeric interaction, was defective in trafficking to the prevacuolar compartment and localized primarily to the trans-Golgi network. In addition, overexpression of C2A, but not wild-type AtVSR1, inhibited trafficking of soluble proteins to the vacuole and caused their secretion into the medium. Furthermore, C2A:hemagglutinin in transgenic plants interfered with the homomeric interaction of endogenous AtVSR1 and inhibited vacuolar trafficking of sporamin:green fluorescent protein. These data suggest that homomeric interaction of AtVSR1 is critical for its function as a vacuolar sorting receptor.Newly synthesized organellar proteins are delivered to their respective organelles by a complex mechanism of transport. Vacuolar or secretory proteins are initially sorted and translocated into the endoplasmic reticulum (ER) cotranslationally (Crowley et al., 1994; Rapoport et al., 1996). After correct folding into a mature protein and assembly into complexes in the ER, these proteins are transported to the Golgi complex by COPII vesicles (Lee et al., 2004; Tang et al., 2005). Proteins that arrive nondiscriminantly to the Golgi complex are subject to sorting primarily at the trans-Golgi network (TGN), and depending on their final destination, they are transported to the prelysosomal or prevacuolar compartment (PVC; Harasaki et al., 2005; Traub, 2005). Lysosomal/vacuolar cargo-sorting receptors play a critical role in the sorting of cargoes at this step (Marcusson et al., 1994; Hadlington and Denecke, 2000; Gu et al., 2001; Tse et al., 2009).In plant cells, the search for vacuolar sorting receptors led to the identification of an 80-kD protein called BP80 (Kirsch et al., 1994, 1996; Paris and Neuhaus, 2002). BP80 is a type I membrane protein and a member of a highly conserved family of proteins in plants termed vacuolar sorting receptors (VSRs; Kirsch et al., 1994, 1996; Ahmed et al., 1997). BP80/VSRs localize primarily to the PVC, with a minor portion located in the TGN (Sanderfoot et al., 1998; Li et al., 2002; Tse et al., 2004). Thus, it has been proposed that BP80/VSRs shuttle between the PVC and the TGN. In the TGN, they are involved in sorting of vacuolar proteins containing a vacuolar sorting motif, NPIR, for packaging into clathrin-coated vesicles (CCVs). In support of this theory, it was shown that in vitro, BP80/VSR binds to the N-terminal propeptide-sorting signal, the NPIR motif (Kirsch et al., 1994, 1996; Ahmed et al., 1997, 2000). In addition, overexpression of the ER-localized luminal domain of PV72, a seed-specific vacuolar sorting receptor, interferes with the transport of an NPIR-containing proteinase in Arabidopsis (Arabidopsis thaliana) leaves (Watanabe et al., 2004). The biological role of BP80/VSRs was demonstrated in protoplasts. Expression of a mutant form of BP80/VSR, in which the luminal domain was replaced with GFP, resulted in secretion of a soluble vacuolar protein, indicating that BP80/VSR functions in protein trafficking to the lytic vacuole (daSilva et al., 2005). In addition, recently it has been demonstrated that AtVSR1 plays a role in trafficking of protein storage vacuoles in plant seed cells (Shimada et al., 2003). In the atvsr1 mutant, storage proteins were secreted into the apoplastic space of Arabidopsis seeds. In this case, the sorting signal recognized by AtVSR1 may be different from the NPIR motif found in proteins destined to the central vacuole.Although there is mounting evidence that BP80/VSR functions as a vacuolar sorting receptor in plant cells (daSilva et al., 2005; Oliviusson et al., 2006), the detailed mechanism of its action remains poorly understood. Man-6-P receptors and Vps10p, the sorting receptors for soluble lysosomal and vacuolar hydrolases in animal and yeast, respectively, recruit adaptor proteins such as adaptor protein complex 1 (AP-1) and Golgi-localized, γ-ear-containing Arf-binding proteins using the C-terminal cytoplasmic domain (CCD; Johnson and Kornfeld, 1992; Dintzis et al., 1994; Honing et al., 1997; Seaman et al., 1997; Nothwehr et al., 2000; Puertollano et al., 2001; Dennes et al., 2002; Doray et al., 2002; Nakatsu and Ohno, 2003). Similarly, the CCD of BP80/VSR may also recruit accessory proteins for CCV formation at the TGN. Indeed, AtVSR1 interacts with EpsinR1 (formally EPSIN1), one of the epsin homologs in Arabidopsis (Song et al., 2006). Since EpsinR1 interacts with clathrin directly, this interaction may play a role in CCV formation. In addition, the CCD of BP80 contains a highly conserved sequence motif, YMPL, which conforms to the consensus sequence motif YXXΦ (where X is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) for binding to AP complexes. A peptide containing the YMPL motif binds in vitro to Arabidopsis μA, a close homolog of AP μ-adaptin in animal cells. The importance of the YXXΦ motif has also been confirmed by a recent study showing that mutation of the YXXΦ motif of BP80 caused its mistargeting in tobacco (Nicotiana tabacum) cells (daSilva et al., 2006). However, the exact role of the YXXΦ motif has not been addressed in trafficking of vacuolar proteins in vivo.In an effort to understand the action mechanism of BP80/VSRs in plant cells, we examined the interaction of AtVSR1 with its binding partners. Here, we demonstrate that AtVSR1 undergoes homomeric interaction through the transmembrane domain (TMD) and CCD and that the homomeric interaction is critical for its function as sorting receptor of vacuolar proteins.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

Autophagy Plays a Role in Chloroplast Degradation during Senescence in Individually Darkened Leaves

Chloroplasts contain approximately 80% of total leaf nitrogen and represent a major source of recycled nitrogen during leaf senescence. While bulk degradation of the cytosol and organelles in plants is mediated by autophagy, its role in chloroplast catabolism is largely unknown. We investigated the effects of autophagy disruption on the number and size of chloroplasts during senescence. When leaves were individually darkened, senescence was promoted similarly in both wild-type Arabidopsis (Arabidopsis thaliana) and in an autophagy-defective mutant, atg4a4b-1. The number and size of chloroplasts decreased in darkened leaves of wild type, while the number remained constant and the size decrease was suppressed in atg4a4b-1. When leaves of transgenic plants expressing stroma-targeted DsRed were individually darkened, a large accumulation of fluorescence in the vacuolar lumen was observed. Chloroplasts exhibiting chlorophyll fluorescence, as well as Rubisco-containing bodies, were also observed in the vacuole. No accumulation of stroma-targeted DsRed, chloroplasts, or Rubisco-containing bodies was observed in the vacuoles of the autophagy-defective mutant. We have succeeded in demonstrating chloroplast autophagy in living cells and provide direct evidence of chloroplast transportation into the vacuole.Chloroplasts contain 75% to 80% of total leaf nitrogen mainly as proteins (Makino and Osmond, 1991). During leaf senescence, chloroplast proteins are gradually degraded as a major source of nitrogen for new growth (Wittenbach, 1978; Friedrich and Huffaker, 1980; Mae et al., 1984), correlating with a decline in photosynthetic activity, while chloroplasts gradually shrink and transform into gerontoplasts, characterized by the disintegration of the thylakoid membranes and accumulation of plastoglobuli (for a recent review, see Krupinska, 2006). Concomitantly, a decline in the cellular population of chloroplasts is also evident in many cases, for example, during natural (Kura-Hotta et al., 1990; Inada et al., 1998), dark-induced (Wittenbach et al., 1982), and nutrient-limited senescence (Mae et al., 1984; Ono et al., 1995), suggesting the existence of a whole chloroplast degradation system. Some electron microscopic studies have shown whole chloroplasts in the central vacuole, which is rich in lytic hydrolases (Wittenbach et al., 1982; Minamikawa et al., 2001). However, there is no direct evidence of chloroplasts moving into the vacuole in living cells and the mechanism of transport is not yet understood (Hörtensteiner and Feller, 2002; Krupinska, 2006).The most abundant chloroplast protein is Rubisco (EC 4.1.1.39), comprising approximately 50% of the soluble protein (Wittenbach, 1978). The amount of Rubisco decreases rapidly in the early phase of leaf senescence, although more slowly in the later phase (Friedrich and Huffaker, 1980; Mae et al., 1984). In contrast, the chloroplast number remains relatively constant, making it impossible to explain Rubisco loss solely by whole chloroplast degradation. However, the mechanism of intrachloroplastic Rubisco degradation is still unknown (for review, see Feller et al., 2008). Using immunoelectron microscopy, we previously demonstrated in naturally senescing wheat (Triticum aestivum) leaves that Rubisco is released from chloroplasts into the cytoplasm and transported to the vacuole for subsequent degradation in small spherical bodies, named Rubisco-containing bodies (RCBs; Chiba et al., 2003). Similar chloroplast-derived structures were also subsequently confirmed in senescent leaves of soybean (Glycine max) and/or Arabidopsis (Arabidopsis thaliana) by electron microscopy (Otegui et al., 2005), and recently in tobacco (Nicotiana tabacum) leaves by immunoelectron microscopy, although the authors gave them a different name, Rubisco vesicular bodies (Prins et al., 2008). RCBs have double membranes, which seem to be derived from the chloroplast envelope; thus, the RCB-mediated degradation of stromal proteins represents a potential mechanism for chloroplast shrinkage during senescence. We recently demonstrated that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole by ATG-dependent autophagy via RCBs, using leaves treated with concanamycin A, a vacuolar H⁺-ATPase inhibitor (Ishida et al., 2008). To investigate further, we wished to observe chloroplast autophagy and degradation directly in living cells to determine whether autophagy is responsible for chloroplast shrinkage and whether it is involved in the vacuolar degradation of whole chloroplasts during leaf senescence.Autophagy is known to be a major system for the bulk degradation of intracellular proteins and organelles in the vacuole in yeast and plants, or the lysosome in animals (for detailed mechanisms, see reviews by Ohsumi, 2001; Levine and Klionsky, 2004; Thompson and Vierstra, 2005; Bassham et al., 2006). In those systems, a portion of the cytoplasm, including entire organelles, is engulfed in membrane-bound vesicles and delivered to the vacuole/lysosome. A recent genome-wide search confirmed that Arabidopsis has many genes homologous to the yeast autophagy genes (ATGs; Doelling et al., 2002; Hanaoka et al., 2002; for detailed functions of ATGs, see the reviews noted above). Using knockout mutants of ATGs and a monitoring system with an autophagy marker, GFP-ATG8, numerous studies have demonstrated the presence of the autophagy system in plants and its importance in several biological processes (Yoshimoto et al., 2004; Liu et al., 2005; Suzuki et al., 2005; Thompson et al., 2005; Xiong et al., 2005, 2007; Fujiki et al., 2007; Phillips et al., 2008). These articles suggest that autophagy plays an important role in nutrient recycling during senescence, especially in nutrient-starved plants. The atg mutants exhibited an accelerated loss of some chloroplast proteins, but not all, under nutrient-starved conditions and during senescence, suggesting that autophagy is not the sole mechanism for the degradation of chloroplast proteins; other, as yet unidentified systems must be responsible for the degradation of chloroplast contents when the ATG system is compromised (Levine and Klionsky, 2004; Bassham et al., 2006). However, it still remains likely that autophagy is responsible for the vacuolar degradation of chloroplasts in wild-type plants.Prolonged observation is generally required to follow leaf senescence events in naturally aging leaves and senescence-associated processes tend to become chaotic over time. To observe chloroplast degradation over a short period, and to draw clear conclusions, a suitable experimental model of leaf senescence is required. Weaver and Amasino (2001) reported that senescence is rapidly induced in individually darkened leaves (IDLs) of Arabidopsis, but retarded in plants subjected to full darkness. In addition, Keech et al. (2007) observed a significant decrease of both the number and size of chloroplasts in IDLs within 6 d.In this study, using IDLs as a senescence model, we aimed to investigate the involvement of autophagy in chloroplast degradation. We show direct evidence for the transport of whole chloroplasts and RCBs to the vacuole by autophagy.     

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as ''lethal exosomes'' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types.¹ ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process.^{2, 3} Two main pathways are involved in MVB maturation.^{4, 5} In addition to the ESCRT (endosomal complex required for traffic) proteins,⁶ there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA),⁷ ceramides⁸ and diacylglycerol (DAG)⁹ contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activation-induced cell death (AICD), antigen presentation and intercellular miRNA exchange.^{10, 11, 12, 13, 14, 15} The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins.^{14, 16, 17, 18, 19, 20, 21} These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes.²² DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG.^{23, 24}The secretory vesicle pathway involves several DAG-controlled checkpoints at which DGKα may act; these include vesicle formation and fission at the trans-Golgi network (TGN), MVB maturation, as well as their transport, docking and fusion to the plasma membrane.^{9, 16, 17, 18, 19, 20} The molecular components that regulate some of these trafficking processes include protein kinase D (PKD) family members.²¹ PKD1 activity, for instance, regulates fission of transport vesicles from TGN via direct interaction with the pre-existing DAG pool at this site.¹⁹ The cytosolic serine/threonine kinases PKD1, PKD2 and PKD3^{(ref. 21)} are expressed in a wide range of cells, with PKD2 the most abundant isotype in T lymphocytes.^{25, 26} PKD have two DAG-binding domains (C1a and C1b) at the N terminus,²¹ which mediate PKD recruitment to cell membranes. Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation.²⁷Based on our previous studies showing DGKα regulation of DAG in MVB formation and exosome secretion,^{9, 14, 28} and the identification of PKD1/2 association to MVB,¹⁴ we hypothesized that DGKα control of DAG mediates these events, at least in part, through PKD. Here we explored whether, in addition to its role in vesicle fission from TGN,¹⁹ PKD regulates other steps in the DAG-controlled secretory traffic pathway. Using PKD-deficient cell models, we analyzed the role of PKD1/2 in MVB formation and function, and demonstrate their implication in exosome secretory traffic.     

Focus on Weed Control: In Vivo 31P-Nuclear Magnetic Resonance Studies of Glyphosate Uptake,Vacuolar Sequestration,and Tonoplast Pump Activity in Glyphosate-Resistant Horseweed

Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. ³¹P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate [AMPA] and N-methyl glyphosate [NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. ³¹P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells.Glyphosate is arguably the world’s most important herbicide (Duke and Powles, 2008). Environmental factors affecting its uptake and translocation in higher plants have been widely studied (Kells and Rieck, 1979; Coupland, 1983; Devine et al., 1983; Masiunas and Weller, 1988; Zhou et al., 2007). Notably, the role of light is important for effective uptake and translocation, suggesting that metabolic energy plays a role in this process (Simarmata et al., 2003; Shaner et al., 2005). Death of the whole plant requires effective glyphosate translocation from source to sink tissue, a process requiring ATP to maintain Suc gradients, which drive phloem movement (Bromilow et al., 1990; Dill et al., 2010).Weed species have developed glyphosate-resistant (GR) biotypes during the past decade (Heap, 2014). This has spurred interest in factors that may contribute to resistant attribute(s) as well as methods that can be used to screen plants for herbicide toxicity (Shaner, 2010). Resistance mechanisms have been reported for horseweed (Conyza canadensis; Feng et al., 2004; Zelaya et al., 2004; Ge et al., 2010, 2011), Palmer amaranth (Amaranthus palmeri; Gaines et al., 2010), and ryegrass (Lolium rigidum and Lolium multiflorum; Powles et al., 1998; Perez et al., 2004; Ge et al., 2012).Since glyphosate is foliar applied, glyphosate toxicity involves a multistep delivery process. Glyphosate must traverse the nonliving structures of the leaf cuticle and the cell walls of the epidermis, apoplast, and mesophyll prior to accessing the phloem for transport to sink tissues (Bromilow et al., 1990; Bromilow and Chamberlain, 2000). Indeed, restriction of glyphosate delivery to the plant cell cytoplasm (and chloroplast) by any means is, in itself, a resistance mechanism (Shaner, 2009; Ge et al., 2013). Elucidation of key factors governing delivery to the intracellular milieu of plant source leaves is critical for developing a complete understanding of the mechanism(s) of resistance to glyphosate.Glyphosate’s phosphono group offers the opportunity to employ in vivo ³¹P-NMR spectroscopy to track glyphosate movement and metabolism, additionally including monitoring of cellular pH, and gradients therein, and ATP levels, both indicators of tissue viability (Roberts, 1984). This laboratory has extended the ³¹P-NMR approach initially used by Gout et al. (1992) with suspension-cultured sycamore (Acer pseudoplatanus) cells. The initial findings, that source and sink leaf tissue from GR horseweed rapidly and avidly sequestered glyphosate within the vacuole compartment and that leaf tissue from glyphosate-sensitive (GS) horseweed did not, led to the hypothesis that vacuole sequestration was a key, perhaps the dominant, component of the resistance mechanism (Ge et al., 2010). It was then shown that GR horseweed acclimated and maintained at cold temperature (approximately 10°C–12°C) did not rapidly and avidly sequester glyphosate within the vacuole. Importantly, under such conditions, GR horseweed succumbed to the toxic effects of glyphosate. In short, by preventing glyphosate sequestration, GR horseweed became glyphosate sensitive, a laboratory finding confirmed in the field (Ge et al., 2011).The proposition that, by limiting the herbicide available for translocation, glyphosate vacuole sequestration could serve as an important if not dominant resistance mechanism was further strengthened by experiments that showed vacuolar glyphosate sequestration correlated with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe (Ge et al., 2012). However, ³¹P-NMR studies of other weeds revealed that in some species, for example, Palmer amaranth, waterhemp (Amaranthus tuberculatus), and johnsongrass (Sorghum halepense), resistance correlated strongly with a lack of glyphosate uptake into the plant cell, a frontline resistance mechanism (Ge et al., 2013).Throughout these previous ³¹P-NMR studies, the finding that plants could regulate the compartmental access of glyphosate led us to speculate that the apoplast, tonoplast, and perhaps chloroplast possessed glyphosate-active transporters whose up-regulation or down-regulation and/or expression would confer resistance (Ge et al., 2010, 2011, 2012, 2013). This hypothesis motivated additional in vivo ³¹P-NMR experiments to further describe the determinants of glyphosate delivery in horseweed leaf tissue. Specifically, experiments with GR horseweed were designed with the goal of probing the transporter hypothesis.Findings from these experiments are reported herein and are consistent with the existence of a tonoplast transporter that is responsible for glyphosate resistance via vacuole sequestration. As described here, vacuole sequestration requires ATP, is active for multiple substrates, and shows substrate competition. Furthermore, glyphosate entry into the cell can be markedly inhibited by vanadate pretreatment. These characteristics are similar to those of ATP-binding cassette transporters in plants (Hetherington et al., 1998; Rea, 2007; Verrier et al., 2008; Prosecka et al., 2009; Conte and Lloyd, 2011) and mammalian cells (van de Ven et al., 2009; Ernst et al., 2010).     

The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

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.