Yonglun Zeng

Organelle pH in the Arabidopsis endomembrane system.

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein (GFP) (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin (plant-solubility-modified ecliptic pHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin (plant-solubility-modified ratiomatric pHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network (TGN) and multivesicular body (MVB) is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H(+)-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

This work identifies the Arabidopsis MON1 and CCZ1 proteins as forming a dimeric complex that functions as a Rab5 effector and a Rab7 GEF to mediate Rab5-to-Rab7 conversion on PVCs. Arabidopsis Rab7 and mon1 mutants contained enlarged PVCs and fragmented vacuoles, and exhibited defects in vacuolar trafficking and retarded-growth phenotypes. Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3fT22N) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.

Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation

Significance Macroautophagy (hereafter as autophagy) involves the delivery of cytosolic materials via autophagosome upon its fusion with the endosome and lysosome/vacuole. The endosomal sorting complex required for transport (ESCRT) machinery is responsible for the formation of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) and the sorting of ubiquitinated membrane cargos into MVB ILVs for degradation. Here, we show that, in addition to regulating MVB biogenesis, the plant-specific ESCRT component FYVE domain protein required for endosomal sorting 1 (FREE1) also plays dual roles in vacuolar protein transport and autophagic degradation. FREE1 directly interacts with a plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 to manipulate the autophagic degradation in plants. Thus, we demonstrate multiple functions of FREE1 and a direct link between the ESCRT machinery and autophagy process in plants. Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

A Unique Plant ESCRT Component, FREE1, Regulates Multivesicular Body Protein Sorting and Plant Growth

Tight control of membrane protein homeostasis by selective degradation is crucial for proper cell signaling and multicellular organismal development. Membrane proteins destined for degradation, such as misfolded proteins or activated receptors, are usually ubiquitinated and sorted into the intraluminal vesicles (ILVs) of prevacuolar compartments/multivesicular bodies (PVCs/MVBs), which then fuse with vacuoles/lysosomes to deliver their contents to the lumen for degradation by luminal proteases. The formation of ILVs and the sorting of ubiquitinated membrane cargoes into them are facilitated by the endosomal sorting complex required for transport (ESCRT) machinery. Plants possess most evolutionarily conserved members of the ESCRT machinery but apparently lack orthologs of ESCRT-0 subunits and the ESCRT-I component Mvb12. Here, we identified a unique plant ESCRT component called FYVE domain protein required for endosomal sorting 1 (FREE1). FREE1 binds to phosphatidylinositol-3-phosphate (PI3P) and ubiquitin and specifically interacts with Vps23 via PTAP-like tetrapeptide motifs to be incorporated into the ESCRT-I complex. Arabidopsis free1 mutant is seedling lethal and defective in the formation of ILVs in MVBs. Consequently, endocytosed plasma membrane (PM) proteins destined for degradation, such as the auxin efflux carrier PIN2, cannot reach the lumen of the vacuole and mislocalize to the tonoplast. Collectively, our findings provide the first functional characterization of a plant FYVE domain protein, which is essential for plant growth via its role as a unique evolutionary ESCRT component for MVB biogenesis and vacuolar sorting of membrane proteins.

PROTEIN S-ACYL TRANSFERASE10 Is Critical for Development and Salt Tolerance in Arabidopsis

S-acylation or palmitoylation, catalyzed by protein S-acyl transferases (PATs), regulates protein targeting and function. This work shows that Arabidopsis PAT10 regulates pleiotropic developmental processes and stress responses. Putative PAT10 substrates include several calcineurin B-like proteins, whose tonoplast association depends on the function of PAT10. Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B–like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins.

Trans -Golgi Network-Located AP1 Gamma Adaptins Mediate Dileucine Motif-Directed Vacuolar Targeting in Arabidopsis

This work demonstrates that TGN-located AP1 complex mediates dileucine motif-directed vacuolar targeting via the interaction with this conserved sorting signal in plant cells. The deficiency of the AP1 gamma adaptins, as well as the mutations of the dileucine residues, resulted in the mislocalization of tonoplast proteins containing the dileucine motif. Membrane proteins on the tonoplast are indispensible for vacuolar functions in plants. However, how these proteins are transported to the vacuole and how they become separated from plasma membrane proteins remain largely unknown. In this study, we used Arabidopsis thaliana vacuolar ion transporter1 (VIT1) as a reporter to study the mechanisms of tonoplast targeting. We showed that VIT1 reached the tonoplast through a pathway involving the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), prevacuolar compartment, and tonoplast. VIT1 contains a putative N-terminal dihydrophobic type ER export signal, and its N terminus has a conserved dileucine motif (EKQTLL), which is responsible for tonoplast targeting. In vitro peptide binding assays with synthetic VIT1 N terminus identified adaptor protein complex-1 (AP1) subunits that interacted with the dileucine motif. A deficiency of AP1 gamma adaptins in Arabidopsis cells caused relocation of tonoplast proteins containing the dileucine motif, such as VIT1 and inositol transporter1, to the plasma membrane. The dileucine motif also effectively rerouted the plasma membrane protein SCAMP1 to the tonoplast. Together with subcellular localization studies showing that AP1 gamma adaptins localize to the TGN, we propose that the AP1 complex on the TGN mediates tonoplast targeting of membrane proteins with the dileucine motif.

Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana

Significance The underlying mechanisms causing the functional diversity of small GTPase Sar1 paralogs in coat protein complex II-mediated protein endoplasmic reticulum (ER) export remain elusive in higher organisms. Arabidopsis contains five Sar1 homologs. In this study, we show that AtSar1a exhibits a distinct localization and effects on ER cargo export in plants through a unique interaction with the COPII coat protein AtSec23a. This specific pairing is required for the distinct function in ER export under ER stress in Arabidopsis. Our results point to a mechanism underlying the functional diversity of COPII paralogs in eukaryotes. Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.

COPII Paralogs in Plants: Functional Redundancy or Diversity?

In eukaryotes, the best-described mechanism of endoplasmic reticulum (ER) export is mediated by coat protein complex II (COPII) vesicles, which comprise five conserved cytosolic components [secretion-associated, Ras-related protein 1 (Sar1), Sec23-24, and Sec13-31]. In higher organisms, multiple paralogs of COPII components are created due to gene duplication. However, the functional diversity of plant COPII subunit isoforms remains largely elusive. Here we summarize and discuss the latest findings derived from studies of various arabidopsis COPII subunit isoforms and their functional diversity. We also put forward testable hypotheses on distinct populations of COPII vesicles performing unique functions in ER export in developmental and stress-related pathways in plants.

How Vacuolar Sorting Receptor Proteins Interact with Their Cargo Proteins: Crystal Structures of Apo and Cargo-Bound Forms of the Protease-Associated Domain from an Arabidopsis Vacuolar Sorting Receptor

This work explores how vacuolar sorting receptor (VSR) recognizes sequence-specific information on cargo proteins for targeting to the vacuole. Elucidation of the structures of the protease-associated domain of a VSR with and without its cognate cargo peptide, along with mutagenesis and functional studies, provides insights into how VSR recognizes its cargo proteins. In plant cells, soluble proteins are directed to vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptors (VSR). To understand how a VSR recognizes its cargo, we present the crystal structures of the protease-associated domain of VSR isoform 1 from Arabidopsis thaliana (VSR1PA) alone and complexed with a cognate peptide containing the barley (Hordeum vulgare) aleurain VSD sequence of 1ADSNPIRPVT10. The crystal structures show that VSR1PA binds the sequence, Ala-Asp-Ser, preceding the NPIR motif. A conserved cargo binding loop, with a consensus sequence of 95RGxCxF100, forms a cradle that accommodates the cargo-peptide. In particular, Arg-95 forms a hydrogen bond to the Ser-3 position of the VSD, and the essential role of Arg-95 and Ser-3 in receptor-cargo interaction was supported by a mutagenesis study. Cargo binding induces conformational changes that are propagated from the cargo binding loop to the C terminus via conserved residues in switch I-IV regions. The resulting 180° swivel motion of the C-terminal tail is stabilized by a hydrogen bond between Glu-24 and His-181. A mutagenesis study showed that these two residues are essential for cargo interaction and trafficking. Based on our structural and functional studies, we present a model of how VSRs recognize their cargos.

Hormone modulates protein dynamics to regulate plant growth

Endosomal traffic in the plant endomembrane system is a fundamental and complex process that controls many essential cellular, developmental, and physiological functions in plants, including cellular polarization, cytokinesis, metal ion homeostasis, pathogen defense, and hormone transport (1). The secretory and endocytic pathways represent two major anterograde protein transport routes for protein delivery into the vacuole in plant cells (Fig. 1 A ). In the secretory pathway, transportation of newly synthesized soluble vacuolar cargo proteins is mediated by the vacuolar sorting receptors (VSRs) (2). After delivery of the soluble cargos into an intermediate compartment, receptors are recycled by the attachment of conserved sorting nexins (SNXs) and the core subunits of retromer complex (VPS26, VPS29, and VPS35) to the membrane. Nevertheless, the precise localization of the SNXs and the retromer subunits, as well as the identity of the organelles from which VSRs are recycled, remains in debate (3, 4). During endocytosis, plasma membrane (PM) proteins are internalized and delivered into the trans -Golgi network (TGN)/early endosomes in plants (5). Ubiquitinated PM proteins are further sorted into the intralumenal vesicles of multivesicular bodies, previously identified as a prevacuolar compartment (6), by the endosomal sorting complex required for transport machinery for vacuolar degradation (7). Alternatively, PM proteins without a ubiquitin tag (or after removal of ubiquitin by a deubiquitinating enzyme) are recycled back to the PM from the TGN or recycling endosome (RE) (1, 8). In plants, numerous PM proteins undergo endocytosis and endosomal recycling, with the PIN-FORMED (PINs) transporters for the plant hormone auxin being the most studied (9). Polarized PM localization of PINs has a profound developmental importance and is tightly regulated by multiple endosomal trafficking routes, including endocytosis, endosomal recycling, and vacuolar degradation. PINs are internalized via clathrin-mediated endocytosis and then recycled back to the PM via the GNOM-positive putative RE … [↵][1]2To whom correspondence should be addressed. Email: ljiang{at}cuhk.edu.hk. [1]: #xref-corresp-1-1