Post-Golgi trafficking of rice storage proteins requires the small GTPase Rab7 activation complex MON1–CCZ1

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kDa glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage prevacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of vacuolar protein sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1–CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5- and Rab7-dependent pathway.

The small GTPases Rab5- and Rab7-dependent pathway is involved in rice storage protein trafficking to vacuoles.     

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole

Background:Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate.Methods:The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined.Results:PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg²⁺ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly.Conclusion:Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum.General significance:This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.     

Auxin-Mediated Ribosomal Biogenesis Regulates Vacuolar Trafficking in Arabidopsis

In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis.     

The Saccharomyces cerevisiae protein Ccz1p interacts with components of the endosomal fusion machinery

The yeast protein Ccz1p is necessary for vacuolar protein trafficking and biogenesis. In a complex with Mon1p, it mediates fusion of transport intermediates with the vacuole membrane by activating the small GTPase Ypt7p. Additionally, genetic data suggest a role of Ccz1p in earlier transport steps, in the Golgi. In a search for further proteins interacting with Ccz1p, we identified the endosomal soluble N -ethylmaleimide-sensitive factor attachment protein receptor Pep12p as an interaction partner of Ccz1p. Combining the ccz1 Δ mutation with deletions of PEP12 or other genes encoding components of the endosomal fusion machinery, VPS21, VPS9 or VPS45 , results in synthetic growth phenotypes. The genes MON1 and YPT7 also interact genetically with PEP12 . These results suggest that the Ccz1p–Mon1p–Ypt7p complex is involved in fusion of transport vesicles to multiple target membranes in yeast cells.     

Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p

Coupling of Rab GTPase activation and SNARE complex assembly during membrane fusion is poorly understood. The homotypic fusion and vacuole protein sorting (HOPS) complex links these two processes: it is an effector for the vacuolar Rab GTPase Ypt7p and is required for vacuolar SNARE complex assembly. We now report that pure, active HOPS complex binds phosphoinositides and the PX domain of the vacuolar SNARE protein Vam7p. These binding interactions support HOPS complex association with the vacuole and explain its enrichment at the same microdomains on docked vacuoles as phosphoinositides, Ypt7p, Vam7p, and the other SNARE proteins. Concentration of the HOPS complex at these microdomains may be a key factor for coupling Rab GTPase activation to SNARE complex assembly.     

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.     

Retromer association with membranes: Plants have their own rules!

The retromer is an endosome-localized complex involved in protein trafficking. To better understand its function and regulation in plants, we recently investigated how Arabidopsis retromer subunits assemble and are targeted to endosomal membranes and highlighted original features compared with mammals. We characterized Arabidopsis vps26 null mutant and showed that it displays severe developmental defaults similar to those observed in vps29 mutant. Here, we go further by describing new phenotypic defects associated with loss of VPS26 function, such as inhibition of lateral root initiation. Recently, we showed that VPS35 subunit plays a crucial role in the recruitment of the plant retromer to endosomes, probably through an interaction with the Rab7 homolog RABG3f. In this work, we now show that contrary to mammals, Arabidopsis Rab5 homologs do not seem to be necessary for the recruitment of the core retromer to endosomal membranes, which highlights a new specificity of the plant retromer.     

Mechanisms Governing the Endosomal Membrane Recruitment of the Core Retromer in Arabidopsis

The retromer complex localizes to endosomal membranes and is involved in protein trafficking. In mammals, it is composed of a dimer of sorting nexins and of the core retromer consisting of vacuolar protein sorting (VPS)26, VPS29, and VPS35. Although homologs of these proteins have been identified in plants, how the plant retromer functions remains elusive. To better understand the role of VPS components in the assembly and function of the core retromer, we characterize here Arabidopsis vps26-null mutants. We show that impaired VPS26 function has a dramatic effect on VPS35 levels and causes severe phenotypic defects similar to those observed in vps29-null mutants. This implies that functions of plant VPS26, VPS29, and VPS35 are tightly linked. Then, by combining live-cell imaging with immunochemical and genetic approaches, we report that VPS35 alone is able to bind to endosomal membranes and plays an essential role in VPS26 and VPS29 membrane recruitment. We also show that the Arabidopsis Rab7 homolog RABG3f participates in the recruitment of the core retromer to the endosomal membrane by interacting with VPS35. Altogether our data provide original information on the molecular interactions that mediate assembly of the core retromer in plants.     

Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein,in mouse testes

Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.     

Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71, Play Antagonistic Roles in the Regulation of Vacuolar Morphology

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 ⁺ exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 ⁺ caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7.     

The AAA‐type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana

The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant‐negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1) under the control of the trichome‐specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant‐negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant‐negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell‐cycle regulation.     

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.     

The KEEP ON GOING Protein of Arabidopsis Regulates Intracellular Protein Trafficking and Is Degraded during Fungal Infection

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.     

Myrosin Cell Development Is Regulated by Endocytosis Machinery and PIN1 Polarity in Leaf Primordia of Arabidopsis thaliana

Myrosin cells, which accumulate myrosinase to produce toxic compounds when they are ruptured by herbivores, form specifically along leaf veins in Arabidopsis thaliana. However, the mechanism underlying this pattern formation is unknown. Here, we show that myrosin cell development requires the endocytosis-mediated polar localization of the auxin-efflux carrier PIN1 in leaf primordia. Defects in the endocytic/vacuolar SNAREs (syp22 and syp22 vti11) enhanced myrosin cell development. The syp22 phenotype was rescued by expressing SYP22 under the control of the PIN1 promoter. Additionally, myrosin cell development was enhanced either by lacking the activator of endocytic/vacuolar RAB5 GTPase (VPS9A) or by PIN1 promoter-driven expression of a dominant-negative form of RAB5 GTPase (ARA7). By contrast, myrosin cell development was not affected by deficiencies of vacuolar trafficking factors, including the vacuolar sorting receptor VSR1 and the retromer components VPS29 and VPS35, suggesting that endocytic pathway rather than vacuolar trafficking pathway is important for myrosin cell development. The phosphomimic PIN1 variant (PIN1-Asp), which is unable to be polarized, caused myrosin cells to form not only along leaf vein but also in the intervein leaf area. We propose that Brassicales plants might arrange myrosin cells near vascular cells in order to protect the flux of nutrients and water via polar PIN1 localization.     

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.     

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.     

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1_(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H⁺-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.     

Subunit E isoform 1 of vacuolar H+-ATPase OsVHA enables post-Golgi trafficking of rice seed storage proteins

Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H⁺-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.

A subunit of the vacuolar H⁺-ATPase regulating endomembrane luminal pH homeostasis plays a fundamental role in post-Golgi trafficking of rice seed storage proteins.     

Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments

Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS) toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN) Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.