Differential Auxin-Transporting Activities of PIN-FORMED Proteins in Arabidopsis Root Hair Cells

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1–PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.Auxin plays a critical role in plant development and growth by forming local concentration gradients. Local auxin gradients, created by the polar cell-to-cell movement of auxin, are implicated in primary axis formation, root meristem patterning, lateral organ formation, and tropic movements of shoots and roots (for recent review, see Vanneste and Friml, 2009). The cell-to-cell movement of auxin is achieved by auxin influx and efflux transporters such as AUXIN-RESISTANT1 (AUX1)/LIKE-AUX1 for influx and PIN-FORMED (PIN) and the P-glycoprotein (PGP) of ABCB (ATP-binding cassette-type transporter subfamily B) for efflux. Since diffusive efflux of the natural auxin indole-3-acetic acid (IAA; pKa = 4.75) is not favorable and PINs are localized in the plasma membrane in a polar manner, PINs act as rate-limiting factors for cellular auxin efflux and polar auxin transport through the plant body. These PINs'' properties explain why representative physiological effects of auxin transport are associated with PINs.Auxin flows from young aerial parts all the way down to the root tip columella in which an auxin maximum is formed for root stem cell maintenance and moves up toward the root differentiation zone through root epidermal cells, where a part of it travels back to the root tip via cortical cells (Blilou et al., 2005). This directional auxin flow is supported by the polar localization of PINs: PIN1, PIN3, and PIN7 at the basal side of stele cells (Friml et al., 2002a, 2002b; Blilou et al., 2005), PIN4 at the basal side in root stem cells (Friml et al., 2002a), and PIN2 at the upper side of root epidermis and at the basal side of the root cortex (Luschnig et al., 1998; Müller et al., 1998). Another interesting aspect of PIN-mediated auxin transport is the dynamics in directionality of auxin flow due to environmental stimuli-directed changes of subcellular PIN polarity, as exemplified for PIN3, whose subcellular localization changes in response to the gravity vector (Friml et al., 2002b).An intriguing question is how different PIN proteins have different subcellular polarities, which might be attributable to PIN-specific molecular properties, cell-type-specific factors, or both. The different PIN subcellular polarities in different cell types seemingly indicate that cell-type-specific factors are involved in polarity. In the case of PIN1, however, both classes of factors appear to affect its subcellular localization because when expressed under the PIN2 promoter, PIN1 localizes to the upper or basal side of root epidermal cells, depending on the GFP insertion site of the protein (Wiśniewska et al., 2006). A recent study demonstrated that the polar targeting of PIN proteins is modulated by phosphorylation/dephosphorylation of the central hydrophilic loop of PINs, which is mediated by PINOID (PID; a Ser/Thr protein kinase)/PP2A phosphatase (Michniewicz et al., 2007). The central hydrophilic domain of PINs might provide the molecule-specific cue for PIN polarity, together with as yet unknown cell-specific factors. Different recycling behaviors of PINs, which show variable sensitivities to brefeldin A (BFA), also imply different molecular characters among PIN species. Most PIN1 proteins are internalized by BFA treatment, whereas considerable amounts of PIN2 remain in the plasma membrane in addition to internal accumulation after BFA treatment. Recycling and basal polar targeting of PIN1 is dependent on the BFA-sensitive guanine nucleotide exchange factor for adenosyl ribosylation factors (ARF GEFs), GNOM, which is the major target of BFA. In contrast, apical targeting and recycling of PIN2 is independent of GNOM and controlled by BFA-resistant ARF GEFs (Geldner et al., 2003; Kleine-Vehn and Friml, 2008).In contrast to their distinct subcellular localizations, the differential auxin-transporting activities of PINs remain to be studied. The divergent primary structures of PIN proteins are not only indicative of differential subcellular polarity, but also would represent their differential catalytic activities for auxin transport. The auxin efflux activities of Arabidopsis (Arabidopsis thaliana) PINs have been demonstrated using Arabidopsis and heterologous systems: PIN1 and PIN5 in Arabidopsis cells (Petrásek et al., 2006; Mravec et al., 2009); PIN2, PIN3, PIN4, PIN6, and PIN7 in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Lee and Cho, 2006; Petrásek et al., 2006; Mravec et al., 2008); PIN1, PIN2, PIN5, and PIN7 in yeast (Saccharomyces cerevisiae) cells (Petrásek et al., 2006; Blakeslee et al., 2007; Mravec et al., 2009; Yang and Murphy, 2009); and PIN1, PIN2, and PIN7 in HeLa cells (Petrásek et al., 2006; Blakeslee et al., 2007). Among the eight Arabidopsis PIN members, PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7, which share a similar molecular structure in terms of the presence of a long central loop (hereafter called long-looped PINs; Fig. 1A; Supplemental Fig. S1), have been shown to catalyze auxin efflux at the cellular level. On the other hand, PIN5 and PIN8 possess a very short putative central loop (hereafter called short-looped PINs). Although PIN5 was recently shown to be localized in the endoplasmic reticulum (ER) and proposed to transport auxin metabolites into the ER lumen, its cellular function regarding its intracellular auxin-transporting activity has not been shown, and the auxin-transporting activity of PIN8 has yet to be demonstrated. In spite of the same transport directionality (auxin efflux) and similar molecular structures, the long-looped PINs exhibit sequence divergence not only in their central loop, but also in certain residues of the transmembrane domains. This structural divergence of long-looped PINs might be indicative of their differential auxin-transporting activities, which have not yet been quantitatively compared.Open in a separate windowFigure 1.Differential activities of PINs in the Arabidopsis root hair. A, Two distinctive PIN groups with different central hydrophilic loop sizes. Topology of PIN proteins was predicted by four different programs as described in Supplemental Figure S1. Numbers above indicate the number of transmembrane helices for each N- and C-terminal region, and numbers below indicate the number of amino acid residues of the central hydrophilic domain. B, Representative root images of control (Cont; Columbia-0) and root-hair-specific PIN-overexpressing (PINox; ProE7:PIN-GFP or ProE7:PIN [−]) plants. Bar = 100 μm for all. C, Root hair lengths of control and PINox plants. Six to 12 independent transgenic lines (average = 8.3), and 42 to 243 roots (average = 86.8) and 336 to 2,187 root hairs (average = 727.8) per construct, were observed for the estimation of root hair length. Data represent means ± se. The root hair lengths of PIN5ox lines were significantly longer than those of the control (P = 0.016 for PIN5ox; P < 0.0001 for PIN5-GFP1ox and PIN5-GFP2ox).To comparatively assess the cytological behaviors and molecular activities of different PIN members, it would be favorable to use a single assay system that provides a consistent cellular environment and enables quantitative estimation of PIN activity. In previous studies, we adopted the root hair single cell system to quantitatively assay auxin-transporting or regulatory activities of PINs, PGPs, AUX1, and PID (Lee and Cho, 2006; Cho et al., 2007a). Root hair growth is proportional to internal auxin levels in the root hair cell. Therefore, auxin efflux inhibits and auxin influx enhances root hair growth (Cho et al., 2007b; Lee and Cho, 2008). In addition, the use of a root-hair-specific promoter (Cho and Cosgrove, 2002; Kim et al., 2006) for expression of auxin transporters enables the transporters'' biological effect to be pinpointed to only the root hair cell, thus excluding probable non-cell-autonomous effects that could be caused by the general expression of auxin transporters.In this study, we expressed five long-looped PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and two short-looped PINs (PIN5 and PIN8) in root hair cells and compared their auxin-transporting activities and cytological dynamics. To directly measure the radiolabeled auxin-transporting activities of PIN5 and PIN8, we used an additional assay system, tobacco suspension cells. Our data revealed that PINs have differential molecular activities and pharmacological responses and that the short-looped and long-looped PINs have different subcellular localizations.     

拟南芥SNARE因子在膜泡运输中的功能

高等植物细胞含有复杂的内膜系统,通过其特有的膜泡运输机制来完成细胞内和细胞间的物质交流。膜泡运输主要包括运输囊泡的出芽、定向移动、拴留和膜融合4个过程。这4个过程受到许多因子的调控,如Coat、SM、Tether、SNARE和Rab蛋白等,其中SNARE因子在膜融合过程中发挥重要功能。SNARE因子是小分子跨膜蛋白,分为定位于运输囊泡上的v-SNARE和定位于靶位膜上的t-SNARE,两类SNARE结合形成SNARE复合体,促进膜融合的发生。SNARE蛋白在调控植物体生长发育以及对外界环境响应等生理过程中起重要作用。该文对模式植物拟南芥（Arabidopsis thaliana）SNARE因子的最新细胞内定位和功能分析等研究进展进行了概述。     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

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.     

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.     

Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis

Directional transport of the phytohormone auxin is established primarily at the point of cellular efflux and is required for the establishment and maintenance of plant polarity. Studies in whole plants and heterologous systems indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins mediate the cellular efflux of natural and synthetic auxins. However, aromatic anion transport resulting from PGP and PIN expression in nonplant systems was also found to lack the high level of substrate specificity seen in planta. Furthermore, previous reports that PGP19 stabilizes PIN1 on the plasma membrane suggested that PIN-PGP interactions might regulate polar auxin efflux. Here, we show that PGP1 and PGP19 colocalized with PIN1 in the shoot apex in Arabidopsis thaliana and with PIN1 and PIN2 in root tissues. Specific PGP-PIN interactions were seen in yeast two-hybrid and coimmunoprecipitation assays. PIN-PGP interactions appeared to enhance transport activity and, to a greater extent, substrate/inhibitor specificities when coexpressed in heterologous systems. By contrast, no interactions between PGPs and the AUXIN1 influx carrier were observed. Phenotypes of pin and pgp mutants suggest discrete functional roles in auxin transport, but pin pgp mutants exhibited phenotypes that are both additive and synergistic. These results suggest that PINs and PGPs characterize coordinated, independent auxin transport mechanisms but also function interactively in a tissue-specific manner.     

Role of SH3 Domain–Containing Proteins in Clathrin-Mediated Vesicle Trafficking in Arabidopsis

A group of plant AtSH3Ps (Arabidopsis thaliana SH3-containing proteins) involved in trafficking of clathrin-coated vesicles was identified from the GenBank database. These proteins contained predicted coiled-coil and Src homology 3 (SH3) domains that are similar to animal and yeast proteins involved in the formation, fission, and uncoating of clathrin-coated vesicles. Subcellular fractionation and immunolocalization studies confirmed the presence of AtSH3P1 in the endomembrane system. In particular, AtSH3P1 was localized on or adjacent to the plasma membrane and its associated vesicles, vesicles of the trans-Golgi network, and the partially coated reticulum. At all of these locations, AtSH3P1 colocalized with clathrin. Functionally, in vitro lipid binding assay demonstrated that AtSH3P1 bound to specific lipid groups known to accumulate at invaginated coated pits or coated vesicles. In addition, immunohistochemical studies and actin binding assays indicated that AtSH3P1 also may regulate vesicle trafficking along the actin cytoskeleton. Yeast complementation studies suggested that AtSH3Ps have similar functions to the yeast Rvs167p protein involved in endocytosis and actin arrangement. A novel interaction between AtSH3P1 and an auxilin-like protein was identified by yeast two-hybrid screening, immunolocalization, and an in vitro binding assay. The interaction was mediated through the SH3 domain of AtSH3P1 and a proline-rich domain of auxilin. The auxilin-like protein stimulated the uncoating of clathrin-coated vesicles by Hsc70, a reaction that appeared to be inhibited in the presence of AtSH3P1. Hence, AtSH3P1 may perform regulatory and/or scaffolding roles during the transition of fission and the uncoating of clathrin-coated vesicles.     

Functional Evolution of Phosphatidylethanolamine Binding Proteins in Soybean and Arabidopsis

Gene duplication provides resources for novel gene functions. Identification of the amino acids responsible for functional conservation and divergence of duplicated genes will strengthen our understanding of their evolutionary course. Here, we conducted a systemic functional investigation of phosphatidylethanolamine binding proteins (PEBPs) in soybean (Glycine max) and Arabidopsis thaliana. Our results demonstrated that after the ancestral duplication, the lineage of the common ancestor of the FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) subfamilies functionally diverged from the MOTHER OF FT AND TFL1 (MFT) subfamily to activate flowering and repress flowering, respectively. They also underwent further specialization after subsequent duplications. Although the functional divergence increased with duplication age, we observed rapid functional divergence for a few pairs of young duplicates in soybean. Association analysis between amino acids and functional variations identified critical amino acid residues that led to functional differences in PEBP members. Using transgenic analysis, we validated a subset of these differences. We report clear experimental evidence for the functional evolution of the PEBPs in the MFT, FT, and TFL1 subfamilies, which predate the origin of angiosperms. Our results highlight the role of amino acid divergence in driving evolutionary novelty after duplication.     

Post-Translational Regulation of AtFER2 Ferritin in Response to Intracellular Iron Trafficking during Fruit Development in Arabidopsis

Ferritins are major players in plant iron homeostasis. Surprisingly, their overexpression in transgenic plants led only to a moderate increase in seed iron content, suggesting the existence of control checkpoints for iron loading and storage in seeds. This work reports the identification of two of these checkpoints. First, measurement of seed metal content during fruit development in Arabidopsis thaliana reveals a similar dynamic of loading for Fe, Mn, Cu, and Zn. The step controlling metal loading into the seed occurs by the regulation of transport from the hull to the seed. Second, metal loading and ferritin abundance were monitored in different genetic backgrounds affected in vacuolar iron transport （AtVIT1, AtNRAMP3, AtNRAMP4） or plastid iron storage （AtFER1 to 4）. This approach revealed （1） a post-translational reg- ulation of ferritin accumulation in seeds, and （2） that ferritin stability depends on the balance of iron allocation between vacuoles and plastids. Thus, the success of ferritin overexpression strategies for iron biofortification, a promising approach to reduce iron-deficiency anemia in developing countries, would strongly benefit from the identification and engineering of mechanisms enabling the translocation of high amounts of iron into seed plastids.     

The Deubiquitinating Enzyme AMSH3 Is Required for Intracellular Trafficking and Vacuole Biogenesis in Arabidopsis thaliana

Ubiquitination, deubiquitination, and the formation of specific ubiquitin chain topologies have been implicated in various cellular processes. Little is known, however, about the role of ubiquitin in the development of cellular organelles. Here, we identify and characterize the deubiquitinating enzyme AMSH3 from Arabidopsis thaliana. AMSH3 hydrolyzes K48- and K63-linked ubiquitin chains in vitro and accumulates both ubiquitin chain types in vivo. amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. Furthermore, AMSH3 is required for efficient endocytosis of the styryl dye FM4-64 and the auxin efflux facilitator PIN2. We thus present evidence for a role of deubiquitination in intracellular trafficking and vacuole biogenesis.     

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.     

Arabidopsis ATG8-INTERACTING PROTEIN1 Is Involved in Autophagy-Dependent Vesicular Trafficking of Plastid Proteins to the Vacuole

Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with plastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from plastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.     

Intracellular Sorting Signals for Sequential Trafficking of Human Cytomegalovirus UL37 Proteins to the Endoplasmic Reticulum and Mitochondria

Human cytomegalovirus UL37 antiapoptotic proteins, including the predominant UL37 exon 1 protein (pUL37x1), traffic sequentially from the endoplasmic reticulum (ER) through the mitochondrion-associated membrane compartment to the mitochondrial outer membrane (OMM), where they inactivate the proapoptotic activity of Bax. We found that widespread mitochondrial distribution occurs within 1 h of pUL37x1 synthesis. The pUL37x1 mitochondrial targeting signal (MTS) spans its first antiapoptotic domain (residues 5 to 34) and consists of a weak hydrophobicity leader (MTSα) and proximal downstream residues (MTSβ). This MTS arrangement of a hydrophobic leader and downstream proximal basic residues is similar to that of the translocase of the OMM 20, Tom20. We examined whether the UL37 MTS functions analogously to Tom20 leader. Surprisingly, lowered hydropathy of the UL37x1 MTSα, predicted to block ER translocation, still allowed dual targeting of mutant to the ER and OMM. However, increased hydropathy of the MTS leader caused exclusion of the UL37x1 high-hydropathy mutant from mitochondrial import. Conversely, UL37 MTSα replacement with the Tom20 leader did not retarget pUL37x1 exclusively to the OMM; rather, the UL37x1-Tom20 chimera retained dual trafficking. Moreover, replacement of the UL37 MTSβ basic residues did not reduce OMM import. Ablation of the MTSα posttranslational modification site or of the downstream MTS proline-rich domain (PRD) increased mitochondrial import. Our results suggest that pUL37x1 sequential ER to mitochondrial trafficking requires a weakly hydrophobic leader and is regulated by MTSβ sequences. Thus, HCMV pUL37x1 uses a mitochondrial importation pathway that is genetically distinguishable from that of known OMM proteins.During infection of permissive cells, the human cytomegalovirus (HCMV) UL37 immediate-early locus encodes multiple UL37 isoforms (4, 11, 16, 22, 24, 25) (Fig. ​(Fig.1A).1A). The predominant isoform, the UL37 exon 1 protein (pUL37x1), or the viral mitochondrial inhibitor of apoptosis (vMIA), is an essential HCMV gene product required for its growth in humans (17) and in cell culture (14, 20, 36, 47). pUL37x1 induces calcium efflux from the endoplasmic reticulum (ER) (40), regulates viral early gene expression (6, 12), disrupts the F-actin cytoskeleton (35, 40), binds and inactivates Bax at the mitochondrial outer membrane (OMM) (5, 32-34), and inhibits mitochondrial serine protease at late times of infection (27).Open in a separate windowFIG. 1.(A) HCMV UL37 isoforms. UL37 proteins share N-terminal UL37x1 MTS, including a moderately hydrophobic MTSα leader (aa 1 to 22, cylinder), MTSβ proximal basic residues (aa 23 to 29, ++++), downstream acidic (aa 81 to 108, —) and basic (aa 134 to 151, +++) domains. The unique C-terminal sequences encoded by UL37 exon 3 contain an N-glycosylation domain (aa 206 to 391, branches) as well as two additional TM domains (aa 178 to 196 and aa 433 to 459, cylinders). The fusion proteins carrying the full length (pUL37x1 wt_1-163) or MTS (wt_1-36-YFP) with C-terminal fluorophores are represented below. The two UL37x1 antiapoptotic domains are also shown (17). (B) Kinetics of pUL37x1 mitochondrial importation. HFFs were cotransfected with plasmids encoding pUL37x1 wt_1-163-YFP and DsRed1-mito (Clontech). After 2 h, anisomycin (70 μM) was added to the medium. After 12 h, the cells were either fixed with 100% methanol (0 min) or washed with 1× PBS and overlaid with fresh, anisomycin-free medium. The cells were incubated for the indicated times before methanol fixation and confocal imaging. The images were obtained by using comparable settings of aperture and laser power. (C) Colocalization of newly synthesized pUL37x1 with a mitochondrial marker. HFFs transiently transfected with pUL37x1 wt_1-163-YFP (green) were treated with anisomycin-containing medium as in panel B for 12 h. Inhibitor-containing medium was removed, and the cells were washed and overlaid with fresh, anisomycin-free medium for 45 min. At that time, 50 nM MitoTracker Red CMXRos (red, Invitrogen) was added to the medium, followed by incubation for 15 min at 37°C, prior to methanol fixation. The cells were then imaged by confocal microscopy. The panels on the left and center are grayscale. The panel on the right is the color merge of both channels. The small insets are enlargements of the indicated region of interest in the cell. (D) UL37x1 MTS is sufficient for mitochondrial import. HFFs were transiently transfected with expression vectors for wt_1-36-YFP and treated with MitoTracker Red (top row) as described above or for wt_1-163-YFP and DsRed1-mito (bottom). Cells were harvested 24 h later and imaged by confocal microscopy. The left and center panels are grayscale. The panels on the right show merged images of both channels.To accomplish their multiple functions in the cell, HCMV UL37 proteins sequentially traffic from the ER to mitochondria (4, 9, 17, 24-26, 45). The amino-terminal UL37x1 antiapoptotic domain serves as a mitochondrial targeting sequence (MTS) (16, 17, 24, 26). UL37 proteins first translocate into the ER, traffic through the mitochondria-associated membrane (MAM) subcompartment of the ER, and then to the OMM (9, 11, 24-26, 45). The MAM is a lipid-rich subdomain of the ER, which directly contacts mitochondria, allowing for the transfer of lipids from the ER to the OMM and the inner mitochondrial membrane (41), and functionally provides microdomains for efficient coupling of ER to mitochondria calcium transfer (37, 42).The HCMV UL37x1 bipartite MTS includes a weakly hydrophobic leader (MTSα, amino acids [aa] 1 to 22) that is required for ER translocation and mitochondrial import, as well as downstream sequences (MTSβ, aa 23 to 34) that are additionally required for its OMM importation (24) (Fig. ​(Fig.2A).2A). The HCMV UL37 MTS is conserved in the homologous primate CMV UL37x1 genes (28).Open in a separate windowFIG. 2.(A) Conservation of UL37x1 MTS among the primate cytomegaloviruses. The sequences of HCMV, chimpanzee CMV (CCMV), rhesus monkey CMV (RhCMV), and African green monkey (AgmCMV) are shown (top). The boxed areas enclose MTSα, the predicted alpha-helical domain, based upon HMMTOP analysis, within each leader. The MTSβ spans downstream residues 23 to 36. The boldfacing and filled circles indicate identity among primate CMV UL37x1 genes. The HCMV UL37x1 hydrophobic leader was mutated to lowered hydrophobicity by replacement of nonconserved residues V4G, L8G, and L14G while maintaining the same length of the TM in the LH mutant (bottom). The predicted hydrophobicity scores (grand average of hydropathicity, GRAVY, Kyte-Doolittle scale) were calculated for the boxed residues of the wt and LH mutant using ProtParam application on the ExPASy Proteomics Server. (B) Colocalization of UL37x1 LH_1-36-YFP with MitoTracker. HFFs transiently transfected with a vector expressing pUL37x1 LH_1-36-YFP for 24 h were treated with 50 nM MitoTracker as described above and imaged by confocal microscopy. Shown on the left and middle panels are the grayscale images, while the panel on the right is the overlay both channels. The small insets are enlargements of the indicated regions of interest. (C) ER translocation and mitochondrial import of pUL37x1 LH_1-36-YFP and LH_1-163-YFP. HeLa cells were transfected with expression vectors of wt_1-36-YFP, LH_1-36-YFP, or YFP vector alone (top) or wt_1-163-YFP, LH_1-163-YFP, or YFP vector alone (bottom). ER and mitochondrial fractions were isolated as described previously (8, 9). (Top) 10 μg (wt_1-36-YFP and YFP vector alone) or 40 μg (LH_1-36-YFP) of each fraction was analyzed by Westerns with anti-GFP (1:200) antibody. (Bottom) 20 μg of each fraction was analyzed by Western analysis with anti-UL37x1 (DC35, 1:2,500) or Grp75 (1:1,000) antibodies.In contrast, most signal-anchored proteins of the OMM are synthesized in the cytosol as precursors with NH₂-terminal sequences that directly target them to mitochondria (31). Signal-anchored OMM proteins, such as the translocase of the OMM subunits, Tom20 and Tom70 (43, 46), are similar in topology to pUL37x1 and the NH₂-terminal cleavage product, pUL37_NH2, of the UL37 glycoprotein (gpUL37) (26). Tom20 and Tom70 are anchored to the OMM by short NH₂-terminal transmembrane (TM) domains with the bulk of the polypeptides exposed to the cytosol in a type I orientation (21). The important structural elements of their signal anchor sequences are (i) moderate hydrophobicity of the TM domain and (ii) positively charged amino acids in its flanking domain (21, 43). Tom20 is targeted from the cytosol to the OMM by a moderately hydrophobic NH₂-terminal leader (score = 1.826) with a minimal requirement for a net basic charge within one to five residues downstream of the leader (21). The juxtaposed basic residues release the Tom20 hydrophobic leader from the ER-targeting signal recognition particle (SRP) and allow for its direct targeting to the OMM. This arrangement of the Tom20 intracellular sorting signals (20, 41) is similar to that of the MTS of pUL37x1 (22), whose leader, while lower in hydropathy (score = 1.289), is nonetheless ER translocated rather than imported from the cytosol directly into the OMM (24, 26).Our studies were undertaken to define the sequence requirements for pUL37x1 sequential targeting to the ER and to the OMM and to determine whether these signals are distinct from those of other OMM proteins. We examined the potential role of conventional OMM targeting signals (leader hydrophobicity and proximal basic residues) as well as sequences conserved in the homologues of primate CMVs. Unpredictably, UL37x1 MTSβ (aa 23 to 36) did not act analogously to the Tom20 mitochondrial targeting leader. Rather, HCMV UL37x1 sequences retargeted the Tom20 hydrophobic leader to sequential ER to OMM import. Moreover, mutation of conventional mitochondrial targeting basic residues did not markedly alter pUL37x1 mitochondrial import. Similarly, UL37x1 lowered hydrophobicity MTSα mutants dually trafficked to the ER and mitochondria. Conversely, pUL37x1 trafficking was altered by increased hydropathy, which effectively blocked mitochondrial import. From these studies, we conclude that weak hydrophobicity of the pUL37x1 MTSα and downstream residues play a role in directing translocation but involve more complex interplay than previously appreciated. Importantly, two previously unrecognized MTS signals, the consensus MTSα posttranslational modification (PTM) site (²¹SY) and a downstream MTSβ proline-rich domain (PRD, aa 33 to 36), regulated pUL37x1 mitochondrial import.(These studies were performed by C.D.W. in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)     

Trafficking of Proteins through Plastid Stromules

Stromules are thin projections from plastids that are generally longer and more abundant on non-green plastids than on chloroplasts. Occasionally stromules can be observed to connect two plastid bodies with one another. However, photobleaching of GFP-labeled plastids and stromules in 2000 demonstrated that plastids do not form a network like the endoplasmic reticulum, resulting in the proposal that stromules have major functions other than transfer of material from one plastid to another. The absence of a network was confirmed in 2012 with the use of a photoconvertible fluorescent protein, but the prior observations of movement of proteins between plastids were challenged. We review published evidence and provide new experiments that demonstrate trafficking of fluorescent protein between plastids as well as movement of proteins within stromules that emanate from a single plastid and discuss the possible function of stromules.Projections from chloroplasts have been reported sporadically in the literature for over a hundred years (reviewed in Gray et al., 2001; Kwok and Hanson, 2004a) and became established as genuine features of plastids when they were observed by the targeting of green fluorescent protein (GFP) to the stromal compartment (Köhler et al., 1997). This study showed that these projections sometimes appeared to connect discrete plastid bodies, and photobleaching experiments demonstrated flow of GFP from one plastid body to another. After GFP in one plastid body was bleached, fluorescence rapidly recovered as a result of flow from GFP from the unbleached plastids. By continuous bleaching of a stromule connecting two plastids, fluorescence was lost from both plastids. This led to the speculation that there could be an interplastid communication system (Köhler et al., 1997). In a follow-up study to test the degree of interplastid connectedness, the term “stromule” was coined to prevent confusion with other tubular structures in the cell (Köhler and Hanson, 2000). The existence of a stromule-based plastid network was ruled out by these experiments, but movement of protein through stromules was confirmed, and it was proposed that stromules might function to facilitate transport of substances in and out of the plastid by increasing surface area and by placing the plastid compartment in close proximity to other organelles or subcellular structures (Köhler and Hanson, 2000). A study by Schattat et al. (2012) confirmed the absence of a plastid network with the use of a photoconvertible fluorescent protein. These authors also describe photoconversion experiments that appear to contradict our prior work demonstrating flow of GFP between two plastid bodies connected by a stromule. Here, we confirm our prior fluorescence recovery after photobleaching (FRAP) results, showing that proteins can move through stromules between individual plastids, and we demonstrate that a red photoconverted protein can also move into a region where photoconversion has not occurred, provided that potentially damaging levels of light are not used during the photoconversion experiment. We review previous studies showing the lack of an interconnected plastid network and consider other functions for stromules, such as facilitating the transport of enzymes and metabolites to and from the plastid to the vicinity of other organelles or regions of the cell.     

拟南芥甲基结合蛋白基因AtMBP11启动子在种子形成和萌发中的功能鉴定

为研究拟南芥甲基结合蛋白基因AtMBP11在种子形成和萌发过程中的调控模式,克隆拟南芥AtMBP11启动子,将其替换植物表达载体pBI121的35S启动子序列,转入拟南芥基因组中.转基因拟南芥后代卡那霉素抗性发生分离,选取具有3∶1分离比的后代自交,产生纯合的具有单拷贝插入的后代.转基因后代GUS染色结果表明,新克隆的MBP启动子控制基因在种子、花药和花粉中高效表达.通过对AtMBP11核心启动子缺失分析表明,G-box元件是主要功能元件.     

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (Aβ) is the major constituent of senile plaques found in the brains of Alzheimer’s disease patients. Aβ is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by β and γ-secretases. Despite the importance of Aβ to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the γ-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.     

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.