David G Robinson

Universität Heidelberg, Heidelburg, Baden-Württemberg, Germany

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Publications (118)725.89 Total impact

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    David G Robinson · Yu Ding · Liwen Jiang
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    ABSTRACT: Unconventional protein secretion (UPS) is a collective term for mechanisms by which cytosolic proteins that lack a signal peptide ("leaderless secretory proteins" (LSPs)) can gain access to the cell exterior. Numerous examples of UPS have been well documented in animal and yeast cells. In contrast, our understanding of the mechanism(s) and function of UPS in plants is very limited. This review evaluates the available literature on this subject. The apparent large numbers of LSPs in the plant secretome suggest that UPS also occurs in plants but is not a proof. Although the direct transport of LSPs across the plant plasma membrane (PM) has not yet been described, it is possible that as in other eukaryotes, exosomes may be released from plant cells through fusion of multivesicular bodies (MVBs) with the PM. In this way, LSPs, but also small RNAs (sRNAs), that are passively taken up from the cytosol into the intraluminal vesicles of MVBs, could reach the apoplast. Another possible mechanism is the recently discovered exocyst-positive organelle (EXPO), a double-membrane-bound compartment, distinct from autophagosomes, which appears to sequester LSPs.
    Full-text · Article · Sep 2015 · Protoplasma
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    ABSTRACT: Autophagosomes are organelles which deliver cytosolic proteins for degradation in vacuole of the cell. In contrast, EXPO (exocyst positive organelles) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension cultures cells and root tissue cells of transgenic Arabidopsis plants expressing either the EXPO marker AtExo70E2-GFP or the autophagosome marker YFP-ATG8e/f, and using specific antibodies against Exo70E2 and ATG8, we have now established that in normally growing cells EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-RABG3f/YFP-VAMP711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner.
    Full-text · Article · Sep 2015 · Plant physiology
  • David G Robinson

    No preview · Article · Jul 2015 · Molecular Plant
  • David G Robinson · Federica Brandizzi · Chris Hawes · Akihiko Nakano
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    ABSTRACT: The endoplasmic reticulum (ER) is the gateway to the secretory pathway in all eukaryotic cells. Its products subsequently pass through the Golgi apparatus on the way to the cell surface (true secretion) or to the lytic compartment of the cell (vacuolar protein transport). In animal cells, the Golgi apparatus is present as a stationary larger order complex near the nucleus, and transport between the cortical ER and the Golgi complex occurs via an intermediate compartment which is transported on microtubules. By contrast, higher plant cells have discrete mobile Golgi stacks that move along the cortical ER, and the intermediate compartment is absent. Although many of the major molecular players involved in ER-Golgi trafficking in mammalian and yeast (Saccharomyces cerevisiae) cells have homologs in higher plants, the narrow interface (less than 500 nm) between the Golgi and the ER, together with the motility factor, makes the identification of the transport vectors responsible for bidirectional traffic between these two organelles much more difficult. Over the years, a controversy has arisen over the two major possibilities by which transfer can occur: through vesicles or direct tubular connections. In this article, four leading plant cell biologists attempted to resolve this issue. Unfortunately, their opinions are so divergent and often opposing that it was not possible to reach a consensus. Thus, we decided to let each tell his or her version individually. The review begins with an article by Federica Brandizzi that provides the necessary molecular background on coat protein complexes in relation to the so-called secretory units model for ER-Golgi transport in highly vacuolated plant cells. The second article, written by Chris Hawes, presents the evidence in favor of tubules. It is followed by an article from David Robinson defending the classical notion that transport occurs via vesicles. The last article, by Akihiko Nakano, introduces the reader to possible alternatives to vesicles or tubules, which are now emerging as a result of exciting new developments in high-resolution light microscopy in yeast. © 2015 American Society of Plant Biologists. All Rights Reserved.
    No preview · Article · Apr 2015 · Plant physiology
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    Yu Ding · David G Robinson · Liwen Jiang

    Preview · Article · Oct 2014 · Current Opinion in Cell Biology
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    ABSTRACT: p24 proteins belong to a family of type I membrane proteins which cycle between ER and Golgi via COPI and COPII vesicles. Current nomenclature classifies them into four subfamilies, although plant p24 proteins belong to either the p24beta or the p24delta subfamilies. Here, we show that Arabidopsis p24δ5/δ9 and HDEL ligands shift the steady-state distribution of the K/HDEL receptor ERD2 from the Golgi to the ER. We also show that p24δ5/δ9 interact directly with ERD2. This interaction requires the GOLD domain in p24δ5 and is much higher at acidic than at neutral pH, consistent with both proteins interacting at the cis-Golgi. In addition, p24δ5 also inhibits the secretion of HDEL ligands, but not constitutive secretion, showing a role of p24δ5 in retrograde Golgi-to-ER transport. Both p24δ5 and ERD2 interact with ARF1 and COPI subunits, mostly at acidic pH, consistent with COPI vesicles being involved in retrograde transport of both proteins. In contrast, both proteins interact with the COPII subunit Sec23 mostly at neutral pH, consistent with this interaction taking place at the ER for anterograde transport to the Golgi apparatus.This article is protected by copyright. All rights reserved.
    No preview · Article · Oct 2014 · The Plant Journal
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    ABSTRACT: Tissue morphogenesis in plants requires communication between cells, a process involving the trafficking of molecules through plasmodesmata (PD). PD conductivity is regulated by endogenous and exogenous signals. However, the underlying signaling mechanisms remain enigmatic. In Arabidopsis, signal transduction mediated by the receptor-like kinase STRUBBELIG (SUB) contributes to inter-cell layer signaling during tissue morphogenesis. Previous analysis has revealed that SUB acts non-cell-autonomously suggesting that SUB controls tissue morphogenesis by participating in the formation or propagation of a downstream mobile signal. A genetic screen identified QUIRKY (QKY), encoding a predicted membrane-anchored C2-domain protein, as a component of SUB signaling. Here, we provide further insight into the role of QKY in this process. We show that like SUB, QKY exhibits non-cell-autonomy when expressed in a tissue-specific manner and that non-autonomy of QKY extends across several cells. In addition, we report on localization studies indicating that QKY and SUB localize to PD but independently of each other. FRET-FLIM analysis suggests that SUB and QKY are in close contact at PD in vivo. We propose a model where SUB and QKY interact at PD to promote tissue morphogenesis, thereby linking RLK-dependent signal transduction and intercellular communication mediated by PD.
    Preview · Article · Sep 2014 · Development
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    David G Robinson

    Preview · Article · Jun 2014 · Plant physiology
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    Yu Ding · David G Robinson · Liwen Jiang
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    ABSTRACT: As in yeast and mammalian cells, novel unconventional protein secretion (UPS) or unconventional membrane trafficking pathways are now known to operate in plants. UPS in plants is generally associated with stress conditions such as pathogen attack, but little is known about its underlying mechanism and function. Here, we present an update on the current knowledge of UPS in the plants in terms of its transport pathways, possible functions and its relationship to autophagy.
    Full-text · Article · Jun 2014 · Current Opinion in Cell Biology
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    ABSTRACT: 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.
    No preview · Article · Apr 2014 · Trends in Plant Science
  • Yu Ding · David G Robinson · Liwen Jiang
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    ABSTRACT: As in yeast and mammalian cells, novel unconventional protein secretion (UPS) or unconventional membrane trafficking pathways are now known to operate in plants. UPS in plants is generally associated with stress conditions such as pathogen attack, but little is known about its underlying mechanism and function. Here, we present an update on the current knowledge of UPS in the plants in terms of its transport pathways, possible functions and its relationship to autophagy.
    No preview · Article · Jan 2014
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    ABSTRACT: In contrast to a single copy of Exo70 in yeast and mammals, the Arabidopsis genome contains 23 paralogs of Exo70 (AtExo70). Using AtExo70E2 and its GFP fusion as probes, we have recently identified a novel double-membrane organelle termed EXPO (exocyst-positive organelle) that mediates an unconventional protein secretion in plant cells. Here we further demonstrate that AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals. By performing transient expression in Arabidopsis protoplasts we have established that a number of exocyst subunits (especially the members of the Sec family) are unable to be recruited to EXPO in the absence of AtExo70E2. The paralog AtExo70A1 is unable to substitute for AtExo70E2 in this regard. FRET and BiFC analyses have confirmed the interaction between AtExo70E2 and Sec6 and Sec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in an animal cell line (HEK293A cells). Electron microscopy confirmed the presence of double membraned EXPO-like structures in HEK293A cells expressing AtExo70E2. Inversely, neither human nor yeast Exo70 homologues are able to cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation.
    Preview · Article · Dec 2013 · Molecular biology of the cell
  • David G Robinson · Peter Pimpl
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    ABSTRACT: Clathrin-coated vesicles (CCVs) are formed at the plasma membrane and act as vectors for endocytosis. They also assemble at the trans-Golgi network (TGN), but their exact function at this organelle is unclear. Recent studies have examined the effects on vacuolar and secretory protein transport of knockout mutations of the adaptor protein 1 (AP1) μ-adaptin subunit AP1M, but these investigations do not clarify the situation. These mutations lead to the abrogation of multiple trafficking pathways at the TGN and cannot be used as evidence in favour of CCVs being agents for receptor-mediated export of vacuolar proteins out of the TGN. This transport process could just as easily occur through the maturation of the TGN into intermediate compartments that subsequently fuse with the vacuole.
    No preview · Article · Nov 2013 · Trends in Plant Science
  • David G Robinson · Peter Pimpl
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    ABSTRACT: In this article we challenge the widely accepted view that receptors for soluble vacuolar proteins (VSRs) bind to their ligands at the trans-Golgi network (TGN) and transport this cargo via clathrin-coated vesicles (CCV) to a multivesicular prevacuolar compartment. This notion, which we term the "classical model" for vacuolar protein sorting, further assumes that low pH in the prevacuolar compartment causes VSR-ligand dissociation, resulting in a retromer-mediated retrieval of the VSRs to the TGN. We have carefully evaluated the literature with respect to morphology and function of the compartments involved, localization of key components of the sorting machinery, and conclude that there is little direct evidence in its favour. Firstly, unlike mammalian cells where the sorting receptor for lysosomal hydrolases recognizes its ligand in the TGN, the available data suggests that in plants VSRs interact with vacuolar cargo ligands already in the endoplasmic reticulum. Secondly, the evidence supporting the packaging of VSR-ligand complexes into CCV at the TGN is not conclusive. Thirdly, the prevacuolar compartment appears to have a pH unsuitable for VSR-ligand dissociation and lacks the retromer core and the sorting nexins needed for VSR recycling. We present an alternative model for protein sorting in the TGN that draws attention to the much overlooked role of Ca(2+) in VSR-ligand interactions and which may possibly also be a factor in the sequestration of secretory proteins.
    No preview · Article · Sep 2013 · Protoplasma
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    ABSTRACT: p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 (p24δ1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP-p24δ9 (p24δ2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)-p24β3 mainly localizes to the Golgi apparatus (as p24β2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24δ9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24β3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the β and δ subfamilies) which are important for their stability and their coupled trafficking at the ER-Golgi interface. Evidence is also provided for a role for p24δ5 in retrograde Golgi-ER transport of the KDEL-receptor ERD2.
    Full-text · Article · Aug 2013 · Journal of Experimental Botany
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    ABSTRACT: Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.
    Full-text · Article · Jul 2013 · International Journal of Molecular Sciences
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    ABSTRACT: New brefeldin A (BFA) analogues were obtained by introducing a variety of substituents at C15. Most of the BFA analogues exhibited significant biological activity. (15R)-Trifluoromethyl-nor-BFA (3), (15R)-vinyl-nor-BFA (5), their epimers 4 and 6 as well as (15S)-ethyl-nor-BFA (2) were prepared from the key building blocks 12 or 24 by Julia-Kocienski olefination with tetrazolyl sulfones and subsequent macrolactonization. The vinyl derivative 5 allowed BFA analogues to be synthesized by hydroboration and Suzuki-Miyaura coupling. The following biological properties were assessed: (a) inhibition of cell growth of human cancer cells (NCI), (b) induction of morphological changes of the Golgi apparatus of plant and mammalian cells, and (c) influence on the replication of the enterovirus CVB3. Furthermore, conformational aspects were studied by X-ray crystal structure analysis and mole¬cular mechanics calculations, including docking of the BFA analogues into the BFA bind¬ing site of an Arf1/Sec7-complex.
    No preview · Article · Jun 2013 · Journal of Medicinal Chemistry
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    Junqi Wang · Jinbo Shen · Yi Cai · David G Robinson · Liwen Jiang
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    ABSTRACT: This study investigated the subcellular location of mung bean (Vigna radiata) 8S globulin in transient expression systems as well as in tobacco (Nicotiana tabacum) BY-2 cells and different tissues from a transgenic Arabidopsis (Arabidopsis thaliana) line stably expressing this storage globulin. When transiently expressed in protoplasts from both BY-2 cells and Arabidopsis suspension cultured cells, the 8S globulin located to structures that were neither Golgi nor pre-vacuolar compartments (PVCs). Immunogold electron microscopy of the transgenics reveals the 8S globulin-positive structures to be small, spherical, ribosome-covered endoplasmic reticulum (ER)-derived bodies. In BY-2 cells and all vegetative cells, the 8S globulin was present as a pro-form. However, in Arabidopsis embryos, with the onset of endogenous storage protein synthesis, the 8S globulin exited the ER and passed through the PVC to the protein storage vacuole where it was processed to its smaller mature form. These results clearly demonstrated that, when taken out of context and expressed in vegetative cells, the mung bean 8S storage globulin cannot exit the ER, and indicate that natural targeting of storage proteins to the vacuole should be better studied in the maturing seed.
    Full-text · Article · Feb 2013 · Journal of Experimental Botany
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    ABSTRACT: ADP-ribosylation factor 1 (Arf1), a member of the small GTP-binding proteins, plays a pivotal role in protein trafficking to multiple organelles. In its GDP-bound form, Arf1 is recruited from the cytosol to organelle membranes, where it functions in vesicle-mediated protein trafficking. However, the mechanism of Arf1-GDP recruitment remains unknown. Here, we provide evidence that two Glo3p-type Arf GTPase activating proteins (ArfGAPs), ArfGAP domain 8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis. RNAi plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNAi plants, Arf1 was poorly recruited to the Golgi apparatus. Conversely, high levels of AGD8 and AGD9 induced Arf1 accumulation at the Golgi and suppressed Golgi disruption and inhibition of vacuolar trafficking that was caused by overexpression of AGD7. Based on these results, we propose that the Glo3p-type ArfGAPs AGD8 and AGD9 recruits Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth.
    No preview · Article · Dec 2012 · Plant physiology
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    ABSTRACT: PDMP (d-l-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol) is a well-known inhibitor of glucosylceramide synthase (GCS), a key enzyme in sphingolipid biosynthesis. Through the resultant increase in ceramides which interact with mTOR and Beclin1 (Atg6), this drug is also known to induce macroautophagy in mammalian cells. This study investigated the response of Arabidopsis root cells to PDMP, and what are probably numerous tightly packed small vacuoles in the control cells appear to fuse to form a single globular-shaped vacuole. However, during this fusion process, cytoplasm channels between the individual vacuoles become trapped in deep invaginations of the tonoplast. In both optical sections in the confocal laser scanning microscope and in ultrathin sections in the electron microscope, these invaginations have the appearance of cytoplasmic inclusions in the vacuole lumen. These changes in vacuole morphology are rapid (occurring within minutes after application of PDMP) and are independent of ongoing protein synthesis. The tonoplast invaginations remain visible for hours, but after 24h almost all disappear. Experiments designed to examine whether ceramide levels might be the cause of the PDMP effect have not proved conclusive. On the other hand, this study has been able to rule out the release of Ca2+ ions from intracellular stores as a contributing factor.
    Full-text · Article · Dec 2012 · Journal of Experimental Botany

Publication Stats

6k Citations
725.89 Total Impact Points

Institutions

  • 2003-2015
    • Universität Heidelberg
      • • Centre of Organismal Studies (COS)
      • • Division of Stem Cell Biology
      Heidelburg, Baden-Württemberg, Germany
  • 2007
    • The University of Hong Kong
      • Department of Biology
      Hong Kong, Hong Kong
  • 2004
    • The Chinese University of Hong Kong
      • Department of Biology
      Hong Kong, Hong Kong
  • 1989-2002
    • Georg-August-Universität Göttingen
      • • Albrecht-von-Haller-Institute for Plant Sciences
      • • Department of Geobiology
      • • Department of Plant Biochemistry
      Göttingen, Lower Saxony, Germany
  • 2000-2001
    • University of Leeds
      • Centre for Plant Sciences
      Leeds, England, United Kingdom
  • 1993-1994
    • Universitätsmedizin Göttingen
      Göttingen, Lower Saxony, Germany