The plant-unique protein DRIF1 coordinates with sorting nexin 1 to regulate membrane protein homeostasis.

Membrane protein homeostasis is fine-tuned by the cellular pathways for vacuolar degradation and recycling, which ultimately facilitate plant growth and cell-environment interactions. The endosomal sorting complex required for transport (ESCRT) machinery plays important roles in regulating intraluminal vesicle (ILV) formation and membrane protein sorting to vacuoles. We previously showed that the plant-specific ESCRT component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) performs multiple functions in plants, although the underlying mechanisms remain elusive. In this study, we performed a suppressor screen of the FREE1-RNAi mutant and identified and characterized 2 suppressor of free1 (sof) mutants in Arabidopsis (Arabidopsis thaliana). These mutants, sof10 and sof641, result in a premature stop codon or a missense mutation in AT5G10370, respectively. This gene was named DEAH and RING domain-containing protein as FREE1 suppressor 1 (DRIF1). DRIF1 has a homologous gene, DRIF2, in the Arabidopsis genome with 95% identity to DRIF1. The embryos of drif1 drif2 mutants arrested at the globular stage and formed enlarged multivesicular bodies (MVBs) with an increased number of ILVs. DRIF1 is a membrane-associated protein that coordinates with retromer component sorting nexin 1 to regulate PIN-FORMED2 recycling to the plasma membrane. Altogether, our data demonstrate that DRIF1 is a unique retromer interactor that orchestrates FREE1-mediated ILV formation of MVBs and vacuolar sorting of membrane proteins for degradation in plants.

An autophagy-inducing stapled peptide induces mitochondria dysfunction and triggers autotic cell death in triple-negative breast cancer.

Autophagy is a lysosome-dependent bulk degradation process essential for cell viability but excessive autophagy leads to a unique form of cell death termed autosis. Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with notable defect in its autophagy process. In previous studies, we developed stapled peptides that specifically targeted the essential autophagy protein Beclin 1 to induce autophagy and promote endolysosomal trafficking. Here we show that one lead peptide Tat-SP4 induced mild increase of autophagy in TNBC cells but showed potent anti-proliferative effect that could not be rescued by inhibitors of programmed cell death pathways. The cell death induced by Tat-SP4 showed typical features of autosis including sustained adherence to the substrate surface, rupture of plasma membrane and effective rescue by digoxin, a cardioglycoside that blocks the Na+/K+ ATPase. Tat-SP4 also induced prominent mitochondria dysfunction including loss of mitochondria membrane potential, elevated mitochondria reactive oxygen species and reduced oxidative phosphorylation. The anti-proliferative effect of Tat-SP4 was confirmed in a TNBC xenograft model. Our study uncovers three notable aspects of autosis. Firstly, autosis can be triggered by moderate increase in autophagy if such increase exceeds the endogenous capacity of the host cells. Secondly, mitochondria may play an essential role in autosis with dysregulated autophagy leading to mitochondria dysfunction to trigger autosis. Lastly, intrinsic autophagy deficiency and quiescent mitochondria bioenergetic profile likely render TNBC cells particularly susceptible to autosis. Our designed peptides like Tat-SP4 may serve as potential therapeutic candidates against TNBC by targeting this vulnerability.

ADP-ribosylation factor D1 modulates Golgi morphology, cell plate formation, and plant growth in Arabidopsis.

ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana.

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Hydrolysis of organophosphorus by diatom purple acid phosphatase and sequential regulation of cell metabolism.

Phosphorus (P) limitation affects phytoplankton growth and population size in aquatic systems, and consequently limits aquatic primary productivity. Plants have evolved a range of metabolic responses to cope with P limitation, such as accumulation of purple acid phosphatases (PAPs) to enhance acquisition of phosphates. However, it remains unknown whether algae have evolved a similar mechanism. In this study, we examined the role of PAPs in the model microalga Phaeodactylum tricornutum. Expression of PAP1 was enhanced in P. tricornutum cells grown on organophosphorus compared to inorganic phosphate. PAP1 overexpression improved cellular growth and biochemical composition in a growth-phase dependent manner. PAP1 promoted growth and photosynthesis during growth phases and reallocated carbon flux towards lipogenesis during the stationary phase. PAP1 was found to be localized in the endoplasmic reticulum and it orchestrated the expression of genes involved in key metabolic pathways and translocation of inorganic P (Pi), thereby improving energy use, reducing equivalents and antioxidant potential. RNAi of PAP1 induced expression of its homolog PAP2, thereby compensating for the Pi scavenging activity of PAP1. Our results demonstrate that PAP1 brings about sequential regulation of metabolism, and provide novel insights into algal phosphorus metabolism and aquatic primary productivity.

Transient Expression of Fluorescent Fusion Proteins in Arabidopsis Protoplasts.

Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES (Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate the major tools and methods for protein localization analysis using super-resolution imaging.

New insights into AtNBR1 as a selective autophagy cargo receptor in Arabidopsis.

Selective autophagy, mediated by cargo receptors and recruiting specific targets to autophagosomes for degradation and recycling, plays an important role in quality control and cellular homeostasis in eukaryotes. The Arabidopsis AtNBR1 shares a similar domain organization with the mammalian autophagic receptors p62 and NBR1. We recently demonstrated that AtNBR1 functions as a selective autophagy receptor for the exocyst component AtExo70E2, a marker for the Exocyst-positive organelle (EXPO), which was achieved via a specific ATG8-AtNBR1-AtExo70E2 interaction in Arabidopsis. Here we further showed that nbr1 CRISPR mutants exhibit an early senescence phenotype under short-day growth conditions, which can be restored by complementation with expression of AtNBR1pro::AtNBR1-GFP in the mutant. Interestingly, in addition to the typical cytosolic and punctate patterns, YFP-AtNBR1 also exhibited a microtubule pattern particularly in the cortical layer. Treatments with the microtubule depolymerizer oryzalin but not the microfilament depolymerizer latrunculin B abolished the microtubule pattern and affected the vacuolar delivery of YFP-AtNBR1 upon autophagy induction. These results indicated that microtubules may be required for AtNBR1 to shuttle its cargos to the vacuole during plant autophagy. The present study thus sheds new light on the recognition and movement pattern of AtNBR1 in selective autophagy in Arabidopsis.

AtNBR1 Is a Selective Autophagic Receptor for AtExo70E2 in Arabidopsis.

Selective autophagy is a subcellular process whereby cytoplasmic materials are selectively sequestered into autophagosomes for subsequent delivery to the vacuole for degradation and recycling. Arabidopsis (Arabidopsis thaliana) NBR1 (next to BRCA1 gene 1 protein; AtNBR1) has been proposed to function as a selective autophagy receptor in plants, whereby AtNBR1 anchors the ubiquitinated targets to autophagosomes for degradation. However, the specific cargos of AtNBR1 remain elusive. We previously showed that Arabidopsis exocyst subunit EXO70 family protein E2 (AtExo70E2), a marker for exocyst-positive organelle (EXPO), colocalized with the autophagosome marker Arabidopsis autophagy-related protein8 (AtATG8) and was delivered to the vacuole for degradation upon autophagic induction. Here, through multiple analyses, we demonstrate that AtNBR1 is a selective receptor for AtExo70E2 during autophagy in Arabidopsis. First, two novel loss-of-function nbr1 CRISPR mutants (nbr1-c1 and nbr1-c2) showed an early-senescence phenotype under short-day growth conditions. Second, during autophagic induction, the vacuolar delivery of AtExo70E2 or EXPO was significantly reduced in nbr1 mutants compared to wild-type plants. Third, biochemical and recruitment assays demonstrated that AtNBR1 specifically interacted and recruited AtExo70E2 or its EXPO to AtATG8-positive autophagosomes in a ubiquitin-associated (UBA)-independent manner during autophagy. Taken together, our data indicate that AtNBR1 functions as a selective receptor in mediating vacuolar delivery of AtExo70E2 or EXPO in a UBA-independent manner in plant autophagy.

Identification and characterization of unconventional membrane protein trafficking regulators in Arabidopsis: A genetic approach.

Proper trafficking and subcellular localization of membrane proteins are essential for plant growth and development. The plant endomembrane system contains several membrane-bound organelles with distinct functions including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN) or early endosome, prevacuolar compartment (PVC) or multivesicular body (MVB) and vacuole. Multiple approaches have been successfully used to identify and study the regulators and components important for signal transduction, growth and development, as well as membrane trafficking in the endomembrane system in plants. These include the homologous characterization of the counterparts in mammals or yeast employing both reverse genetic as well as the forward genetic screen approaches. However, the deletion or mutation of membrane trafficking related proteins usually leads to seedling lethality due to their essential roles in plant development and organelle biogenesis. To overcome the limitation of lethal phenotype of the target proteins, we used DEX-inducible RNAi knock-down lines to study their function in plants. More recently, we developed and used both RNAi knock-down and T-DNA insertional lines as starting materials to screen for mutations that could suppress and rescue the lethal phenotype, or a suppressor screening. Further characterization of the newly identified suppressor mutants has resulted in the identification of novel negative regulators in mediating membrane trafficking and organelle biogenesis in plants. In this review, we summarize the current approaches in studying protein trafficking in the endomembrane system. We then describe three examples of suppressor screening with distinct starting materials (i.e. FREE1, MON1, and SH3P2 that are regulators of MVB, vacuole, and autophagosomes, respectively) to discuss the rationale, procedures, advantages and disadvantages, and possible outcomes of such a suppressor screening. We finally propose that these novel screening approaches will lead to the identification of new unconventional players in regulating protein trafficking and organelle biogenesis in plants and discuss their impact on plant cell biology research.

Membrane Contact Sites and Organelles Interaction in Plant Autophagy.

Autophagy is an intracellular trafficking and degradation system for recycling of damaged organelles, mis-folded proteins and cytoplasmic constituents. Autophagy can be divided into non-selective autophagy and selective autophagy according to the cargo specification. Key to the process is the timely formation of the autophagosome, a double-membrane structure which is responsible for the delivery of damaged organelles and proteins to lysosomes or vacuoles for their turnover. Autophagosomes are formed by the closure of cup-shaped phagophore which depends on the proper communication with membrane contributors. The endoplasmic reticulum (ER) is a major membrane source for autophagosome biogenesis whereby the ER connects with phagophore through membrane contact sites (MCSs). MCSs are closely apposed domains between organelle membranes where lipids and signals are exchanged. Lipid transfer proteins (LTPs) are a large family of proteins including Oxysterol-binding protein related proteins (ORP) which can be found at MCSs and mediate lipid transfer in mammals and yeast. In addition, interaction between autophagosomes and other organelles can also be detected in selective autophagy for selection and degradation of various damaged organelles. Selective autophagy is mediated by the binding of a receptor or an adaptor between a cargo and an autophagosome. Here we summarize what we know about the MCS between autophagosomes and other organelles in eukaryotes. We then discuss progress in our understanding about ORPs at MCSs in plants and the underlying mechanisms of selective autophagy in plants with a focus on receptors/adaptors that are involved in the interaction of the autophagosome with other cytoplasmic constituents, including the Neighbor of BRCA1 gene 1 (NBR1), ATG8-interacting protein 1 (ATI1), Regulatory Particle Non-ATPase 10 (RPN10), and Dominant Suppressor of KAR2 (DSK2).

Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization.

Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell-cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading.

Signal motif-dependent ER export of the Qc-SNARE BET12 interacts with MEMB12 and affects PR1 trafficking in Arabidopsis.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are well-known for their role in controlling membrane fusion, the final, but crucial step, in vesicular transport in eukaryotes. SNARE proteins contribute to various biological processes including pathogen defense and channel activity regulation, as well as plant growth and development. Precise targeting of SNARE proteins to destined compartments is a prerequisite for their proper functioning. However, the underlying mechanism(s) for SNARE targeting in plants remains obscure. Here, we investigate the targeting mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is involved in protein trafficking in the early secretory pathway. Two distinct signal motifs that are required for efficient BET12 ER export were identified. Pulldown assays and in vivo imaging implicated that both the COPI and COPII pathways were required for BET12 targeting. Further studies using an ER-export-defective form of BET12 revealed that the Golgi-localized Qb-SNARE MEMB12, a negative regulator of pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its interacting partner. Ectopic expression of BET12 caused no inhibition in the general ER-Golgi anterograde transport but caused intracellular accumulation of PR1, suggesting that BET12 has a regulatory role in PR1 trafficking in A. thaliana.