Caiji Gao

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.

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

This work identifies SH3P2 as a novel regulator of autophagy and provides a conserved model for autophagosome biogenesis in Arabidopsis. Autophagosome-related structures, such as isolation membranes and ER-derived omegasome-like structures, are characterized and SH3P2 is shown to bind to PI3P and regulate autophagosome formation via the association with the PI3K complex and ATG8. Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis.

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.

The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic Reticulum Export and Golgi Retention Signals at Its C Terminus

A Golgi-localized polytopic integral membrane protein, EMP12, was shown to contain an endoplasmic reticulum export signal (FVY) and a Golgi retention signal (KXE/D) that interact with COPII and COPI subunits, respectively, in Arabidopsis cells. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells. Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.

Essential role for TrpC5-containing extracellular vesicles in breast cancer with chemotherapeutic resistance

Significance A critical challenge for chemotherapy is development of chemoresistance, but underlying molecular mechanisms remain unclear. In this study, we found that drug-resistant adriamycin-resistant human breast cancer cells possessed numerous transient receptor potential channel 5 (TrpC5) -containing extracellular vesicles (EVs) on the cell surface. Suppressing TrpC5 expression diminished the formation of EVs. Incubation of drug-sensitive recipient cells with EVs endowed recipients with drug-resistant properties. In both human samples and a mouse model of breast cancer, the expression of TrpC5 proteins was high in the tumor, and the levels of TrpC5-positive EVs were high in the circulation. These data suggest a critical role of TrpC5-containing EVs in the transfer of drug resistance. In the future, monitoring TrpC5-containing EVs in the circulation could potentially be used to predict the clinical outcome of chemotherapy. A critical challenge for chemotherapy is the development of chemoresistance in breast cancer. However, the underlying mechanisms and validated predictors remain unclear. Extracellular vesicles (EVs) have gained attention as potential means for cancer cells to share intracellular contents. In adriamycin-resistant human breast cancer cells (MCF-7/ADM), we analyzed the role of transient receptor potential channel 5 (TrpC5) in EV formation and transfer as well as the diagnostic implications. Up-regulated TrpC5, accumulated in EVs, is responsible for EV formation and trapping of adriamycin (ADM) in EVs. EV-mediated intercellular transfer of TrpC5 allowed recipient cells to acquire TrpC5, consequently stimulating multidrug efflux transporter P-glycoprotein production through a Ca2+- and activated T-cells isoform c3-mediated mechanism and thus, conferring chemoresistance on nonresistant cells. TrpC5-containing circulating EVs were detected in nude mice bearing MCF-7/ADM tumor xenografts, and the level was lower after TrpC5–siRNA treatment. In breast cancer patients who underwent chemotherapy, TrpC5 expression in the tumor was significantly higher in patients with progressive or stable disease than in patients with a partial or complete response. TrpC5-containing circulating EVs were found in peripheral blood from patients who underwent chemotherapy but not patients without chemotherapy. Taken together, we found that TrpC5-containing circulating EVs may transfer chemoresistance property to nonchemoresistant recipient cells. It may be worthwhile to further explore the potential of using TrpC5-containing EVs as a diagnostic biomarker for chemoresistant breast cancer.

Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER―Golgi―TGN―PM pathway

How polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway.

Retention mechanisms for ER and Golgi membrane proteins

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins.

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

An Arabidopsis trafficking protein complex is required in the scission of internal vesicles and membrane cargo degradation from both secretory and endocytic pathways. We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1.

Vacuolar Degradation of Two Integral Plasma Membrane Proteins, AtLRR84A and OsSCAMP1, Is Cargo Ubiquitination‐Independent and Prevacuolar Compartment‐Mediated in Plant Cells

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination‐independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine‐rich repeat receptor‐like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans‐Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY‐2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination‐independent manner. Such MVB/PVC‐mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination‐independent and PVC‐mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.