N‐linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis

Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post‐translationally modified by the attachment of N‐glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex‐type N‐glycans, which are located in the N‐terminal ‘PA domain’, the central region and the C‐terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N‐glycans do not affect the targeting of AtVSR1 to pre‐vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N‐glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N‐glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.     

Changes in Calcium/Calmodulin Level and Mitochondrial Membrane Potential in Cochlear Neurons of Newborn Mice Infected with Murine Cytomegalovirus

We sought to elucidate the pathogenesis of hearing loss in newborns due to congenital cytomegalovirus. We used the model of murine cytomegalovirus (MCMV) infection and evaluated concentrations of free calcium, calmodulin levels, and mitochondrial membrane potential in cochlear neurons of infected newborn mice. MCMV infection was established by intracranial inoculation of newborn mice with viral suspension (20 μl of MCMV TCID₅₀—10⁴ IU/0.1 ml); the mice in control group were injected 0.9 % NaCl. Concentration of intracellular free calcium concentration ([Ca²⁺] _i ), mitochondrial membrane potential, and the mRNA level of calmodulin (CaM) in the cochlear neurons were evaluated, when the mice were 1 month old. Compared with control group, intracellular [Ca²⁺] _i and CaM mRNA levels significantly (p < 0.05; both comparisons) increased, while the mitochondrial membrane potential significantly (p < 0.05) decreased in the MCMV-infected group. In conclusion, alteration of [Ca²⁺] _i and CaM levels and mitochondrial membrane potentials in cochlear neurons may be the pathological basis of sensorineural hearing loss associated with MCMV infection.     

Sorting out the role of reactive oxygen species during plant programmed cell death induced by ultraviolet-C overexposure

Previous studies have reported that light is required for activating Arabidopsis programmed cell death (PCD) induced by ultraviolet-C (UV-C) overexposure, and a caspase-like protease cleaving the caspase-3 substrate Asp-Glu-Val-Asp (DEVDase activity) is induced during this process. Our recent report has suggested that a quick burst of reactive oxygen species (ROS), which is mainly derived from mitochondria and chloroplasts, is induced in a light dependent manner during the early stages of UV-induced plant PCD. Concomitantly, the mitochondria undergo serious dysfunction including the MTP loss and the changes in distribution and mobility, which ultimately lead to apoptotic-cell death. Though some of signaling molecules have been elucidated in this type of plant cell death, the molecular mechanism about UV-induce Arabidopsis PCD is still poorly understood when comparing with the study of signaling pathways involved in animal cell apoptosis induced by UV. By using the Arabidopsis mesophyll protoplasts as a reference model, we have begun to shed light on the complexity of signaling pathway in UV-induced plant PCD. Recently we have tried to real-time detect the presence of caspase-like proteolytic activation, and to sort out the key role of ROS as well as to further assess the relationship between the ROS production and caspase-like activation in this type of plant apoptotic cell death.Key words: caspase-like activation, FRET, programmed cell death, reactive oxygen species, ultraviolet-CUltraviolet-C has been shown to be a very convenient trigger to induce PCD in plants and protoplasts.^1,2 Others have shown that UV induction of plant PCD requires light and that caspase-like proteolytic activation is induced in this process.¹ Our recent works have shown that ROS mainly localizing in mitochondria and chloroplasts are produced in a light dependent manner during the early stages of UV stress, and that ROS production and mitochondrial dysfunction play important roles during UV-induced Arabidopsis PCD (Fig. 1).² We also found that if the Arabidopsis plants, which were kept at light for 1 h after UV irradiation then were moved to the dark and kept for 60 h, showed no evident plant death phenomena (unpublished data), though burst of ROS has appeared after UV exposure and subsequent 1 h light irradiation.² In contrast, seedlings developed an obvious bleaching when kept in light for 60 h after UV treatment. These findings prompt us to carry out further investigations to dig out the role of ROS in the execution of this type of cell death, and to ask whether the produced ROS in the early stages is involved in the activation of caspase-like protease.Open in a separate windowFigure 1Hypothetical model of the signal transduction pathways in the plant programmed cell death induced by UV-C overexposure. After UV and light treatment a quick burst of ROS appear in the region of mitochondria and chloroplasts, then the mitochondria undergo functional dysfunction, which ultimately leads to cell death. Caspase-like activation and nucleus damage are also involved in the control of this type cell death. Solid line means the issues have been detected. Dotted line and question marks indicate events that have not been detected in this process. For detailed explanation, see the text.It has been reported that ROS is required for the release of cytochrome c (cyt c) and subsequent activation of caspase-like proteases during heat-shock induced plant PCD, and the addition of caspase inhibitors (zVAD-fmk or AC-DEVD-CHO) can prevent the degradation of cyt c and protect the plant cells from cell death.³ Thus these findings suggest that ROS can trigger the release of cyt c, but do not cause cell death, which requires caspase-like activation.³ Conversely, caspase inhibitors have also shown to effectively block the oxidative burst and the plant cell death induced by camptothecin incubation.⁴ These studies suggest the complex relationship between ROS production and caspase activation during execution of plant PCD event. The ROS production and the mitochondrial dysfunction during UV-induced plant PCD have been illustrated in our research. We have suggested the occurrence of MTP disruption during UV stress; however, whether cyt c is released from mitochondria has not been assessed (Fig. 1). The important roles of cyt c release and subsequent caspase activation have been suggested in various types of programmed cell death including mammal and plant cells.^3,5,6 It will be a very challenging work to detect whether cyt c is released from mitochondria and is involved in the caspase-like proteolytic activation, and to further elucidate the relationship between ROS production and caspase-like activation in UV-induced plant PCD (Fig. 1).The involvement of caspase-like proteases in the control of cell death activation in plants has been shown in various forms of plant PCD.⁷ Using synthetic fluorogenic caspase-3 substrate, DEVD cleavage activity was detected during UV or heat shock-induced apoptosis of plant cells, and caspase inhibitors were able to suppress these types of cell death.^1,3 Caspase-like activities have also been detected in plant hypersensitive response (HR) triggered by tobacco mosaic virus (TMV), or plant PCD induced by chemicals like camptothecin.^8,9 All these experiments suggest the existence of functional caspase proteolytic activity in plant cells undergoing PCD. However, most of these results are from in vitro analysis using synthetic fluorogenic substrates or synthetic peptide inhibitor to caspases, this demand us to further dig out the plant caspase encoding gene and to real-time detect the caspase-like activity in vivo.Another of our ongoing work is aiming to detect the caspase-3-like proteolytic activation in living plant cells during UV-induced plant PCD, which is achieved by using the fluorescence resonance energy transfer (FRET) technique. FRET is the phenomenon whereby a fluorescent molecule—the donor—transfers energy by a nonradiative (through space) mechanism to a neighboring chromophore - the acceptor.¹⁰ FRET as a powerful technique to monitor compartmentation and subcellular targeting as well as to visualize protein-protein interactions and proteases activity in living cells has gained increasing importance for biotechnological applications during the last few years.¹¹ During the past few years FRET technique has been successfully used to monitor interactions and distances between molecules in living plant cells.^12–14 Presently, we have constructed a recombinant caspase substrate to monitor caspase-3-like protease activation in single living plant protoplast in real time. This recombinant is composed of enhanced cyan fluorescence protein (ECFP) as the FRET donor and enhanced yellow fluorescence protein (EYFP) as the acceptor, linked by peptides containing the caspase-3 cleavage sequence, DEVD (ECFP-DEVD-EYFP) as the papers demonstrated. 15 Arabidopsis mesophyll protoplasts have been successfully transiently transfected with our recombinant plasmid for expression of ECFP-DEVD-EYFP fusion proteins under control of the CaMV 35S promoter according to a modified procedure (as described previously, ref. ¹⁶). Preliminary experimental results have proved the feasibility of this method to real-time detect the caspase-like activation in living plant cells during UV-induced plant PCD.Using this FRET probe, we may real-time detect the caspase-like activation during UV-induced plant PCD, and elucidate the relationship between ROS production and caspase-like activation as well as verify our hypothesis that whether ROS is necessary for the activation of caspase-like proteases during this process. So the role of ROS in the execution of this type cell death can be further investigated. These subsequent researches will certainly increase our knowledge about the signal transduction pathways in UV-induced Arabidopsis PCD.     

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.     

基于TurboID的植物蛋白邻近标记实验方法

邻近标记作为近些年发展起来的一项检测活细胞内蛋白互作关系和亚细胞结构蛋白组的新型技术, 已成功应用于多种动植物体系的研究。该技术通过给诱饵蛋白融合一个具有特定催化连接活性的酶, 在酶的催化作用下将小分子底物(如生物素)共价连接到酶邻近的内源蛋白, 通过富集和分析被标记的蛋白可获得与诱饵互作的蛋白组。经定向进化产生的生物素连接酶TurboID具有无蛋白毒性及催化效率高的优势。利用TurboID介导的邻近标记技术分析感兴趣蛋白的邻近蛋白组, 可研究细胞内瞬时发生或微弱的蛋白互作网络, 进而解析复杂的生物学过程。该文详细描述了在拟南芥(Arabidopsis thaliana)中基于TurboID的邻近标记实验方法及注意事项, 旨在为利用这一新技术研究植物蛋白互作关系提供参考。     

Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms

We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

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.     

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.     

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

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.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.     

Protein trafficking in plant cells: Tools and markers

Eukaryotic cells consist of numerous membrane-bound organelles,which compartmentalize cellular materials to fulfil a variety of vital functions.In the post-genomic era,it is widely recognized that identification of the subcellular organelle localization and transport mechanisms of the encoded proteins are necessary for a fundamental understanding of their biological functions and the organization of cellular activity.Multiple experimental approaches are now available to determine the subcellular localizations and dynamics of proteins.In this review,we provide an overview of the current methods and organelle markers for protein subcellular localization and trafficking studies in plants,with a focus on the organelles of the endomembrane system.We also discuss the limitations of each method in terms of protein colocalization studies.