Henri Batoko

A Rab1 GTPase Is Required for Transport between the Endoplasmic Reticulum and Golgi Apparatus and for Normal Golgi Movement in Plants

We describe a green fluorescent protein (GFP)–based assay for investigating membrane traffic on the secretory pathway in plants. Expression of AtRab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTPase AtRab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscent of the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type AtRab1b but not AtRab8c. When a Golgi-targeted and N-glycosylated variant of GFP was coexpressed with AtRab1b(N121I), the variant also accumulated in a reticulate network and an endoglycosidase H–sensitive population appeared. Unexpectedly, expression of AtRab1b(N121I), but not of the wild-type AtRab1b, resulted in a reduction or cessation of vectorial Golgi movement, an effect that was reversed by coexpression of the wild type. We conclude that AtRab1b function is required for transport from the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control of Golgi movement.

FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient, Pf. We previously showed that, in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;1 interact to increase the cell Pf. Here, we report the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells. ZmPIPs were fused to monomeric yellow fluorescent protein and/or monomeric cyan fluorescent protein and expressed transiently in maize mesophyll protoplasts. When expressed alone, ZmPIP1 fusion proteins were retained in the endoplasmic reticulum, whereas ZmPIP2s were found in the plasma membrane. Interestingly, when coexpressed with ZmPIP2s, ZmPIP1s were relocalized to the plasma membrane. Using FRET/fluorescence lifetime imaging microscopy, we demonstrated that this relocalization results from interaction between ZmPIP1s and ZmPIP2s. Immunoprecipitation experiments provided additional evidence for the association of ZmPIP1;2 and ZmPIP2;1 in maize roots and suspension cells. These data suggest that PIP1–PIP2 interaction is required for in planta PIP1 trafficking to the plasma membrane to modulate plasma membrane permeability.

The Arabidopsis Multistress Regulator TSPO Is a Heme Binding Membrane Protein and a Potential Scavenger of Porphyrins via an Autophagy-Dependent Degradation Mechanism

This study establishes that plant TSPO, an enigmatic stress-induced membrane protein, binds porphyrins, including heme in vitro and in vivo, and that heme binding regulates TSPO degradation through autophagy. This work indicates that TSPO expression may, at least in part, transiently protect plant cell from porphyrin-induced damages. TSPO, a stress-induced, posttranslationally regulated, early secretory pathway-localized plant cell membrane protein, belongs to the TspO/MBR family of regulatory proteins, which can bind porphyrins. This work finds that boosting tetrapyrrole biosynthesis enhanced TSPO degradation in Arabidopsis thaliana and that TSPO could bind heme in vitro and in vivo. This binding required the His residue at position 91 (H91), but not that at position 115 (H115). The H91A and double H91A/H115A substitutions stabilized TSPO and rendered the protein insensitive to heme-regulated degradation, suggesting that heme binding regulates At-TSPO degradation. TSPO degradation was inhibited in the autophagy-defective atg5 mutant and was sensitive to inhibitors of type III phosphoinositide 3-kinases, which regulate autophagy in eukaryotic cells. Mutation of the two Tyr residues in a putative ubiquitin-like ATG8 interacting motif of At-TSPO did not affect heme binding in vitro but stabilized the protein in vivo, suggesting that downregulation of At-TSPO requires an active autophagy pathway, in addition to heme. Abscisic acid–dependent TSPO induction was accompanied by an increase in unbound heme levels, and downregulation of TSPO coincided with the return to steady state levels of unbound heme, suggesting that a physiological consequence of active TSPO downregulation may be heme scavenging. In addition, overexpression of TSPO attenuated aminolevulinic acid–induced porphyria in plant cells. Taken together, these data support a role for TSPO in porphyrin binding and scavenging during stress in plants.

A Rab-E GTPase Mutant Acts Downstream of the Rab-D Subclass in Biosynthetic Membrane Traffic to the Plasma Membrane in Tobacco Leaf Epidermis

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1d[NI]) of Arabidopsis thaliana RAB-E1d and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1d[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1d[NI] caused less intracellular secGFP accumulation than RAB-D2a[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2a[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1d[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)–tagged Arabidopsis RAB-F2b. Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1d[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1d, YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1d[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

The Arabidopsis TSPO‐related protein is a stress and abscisic acid‐regulated, endoplasmic reticulum–Golgi‐localized membrane protein

The Arabidopsis gene At2g47770 encodes a membrane-bound protein designated AtTSPO (Arabidopsis thaliana TSPO-related). AtTSPO is related to the bacterial outer membrane tryptophan-rich sensory protein (TspO) and the mammalian mitochondrial 18-kDa translocator protein (18 kDa TSPO), members of the group of TspO/MBR domain-containing membrane proteins. In this study we show that AtTSPO is mainly detected in dry seeds, but can be induced in vegetative tissues by osmotic or salt stress or abscisic acid (ABA) treatment, corroborating available transcriptome data. Using subcellular fractionation, immunocytochemistry and fluorescent protein tagging approaches we present evidence that AtTSPO is targeted to the secretory pathway in plants. Induced or constitutively expressed AtTSPO can be detected in the endoplasmic reticulum and the Golgi stacks of plant cells. AtTSPO tagged with fluorescent protein in transgenic plants (Arabidopsis and tobacco) was mainly detected in the Golgi stacks of leaf epidermal cells. Constitutive expression of AtTSPO resulted in increased sensitivity to NaCl, but not to osmotic stress, and in reduced greening of cultured Arabidopsis cells under light growing conditions. Transgenic Arabidopsis plants overexpressing AtTSPO were more sensitive to ABA-induced growth inhibition, indicating that constitutive expression of AtTSPO may enhance ABA sensitivity. AtTSPO is rapidly downregulated during seed imbibition, and the ABA-dependent induction in plant is transient. Downregulation of AtTSPO seems to be boosted by treatment with aminolevulinic acid. Taken together, these results suggest that AtTSPO is a highly regulated protein, induced by abiotic stress to modulate, at least in part, transient intracellular ABA-dependent stress perception and/or signalling.

Targeting of a Nicotiana plumbaginifolia H+-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+-ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane–span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

The Arabidopsis Abiotic Stress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2;7 through Protein-Protein Interactions and Autophagic Degradation

Expression of a plasma membrane aquaporin is regulated by an abiotic stress-induced protein via intracellular protein-protein interactions and targeting of the aquaporin for autophagic degradation. The Arabidopsis thaliana multi-stress regulator TSPO is transiently induced by abiotic stresses. The final destination of this polytopic membrane protein is the Golgi apparatus, where its accumulation is strictly regulated, and TSPO is downregulated through a selective autophagic pathway. TSPO-related proteins regulate the physiology of the cell by generating functional protein complexes. A split-ubiquitin screen for potential TSPO interacting partners uncovered a plasma membrane aquaporin, PIP2;7. Pull-down assays and fluorescence imaging approaches revealed that TSPO physically interacts with PIP2;7 at the endoplasmic reticulum and Golgi membranes in planta. Intriguingly, constitutive expression of fluorescently tagged PIP2;7 in TSPO-overexpressing transgenic lines resulted in patchy distribution of the fluorescence, reminiscent of the pattern of constitutively expressed yellow fluorescent protein-TSPO in Arabidopsis. Mutational stabilization of TSPO or pharmacological inhibition of the autophagic pathway affected concomitantly the detected levels of PIP2;7, suggesting that the complex containing both proteins is degraded through the autophagic pathway. Coexpression of TSPO and PIP2;7 resulted in decreased levels of PIP2;7 in the plasma membrane and abolished the membrane water permeability mediated by transgenic PIP2;7. Taken together, these data support a physiological role for TSPO in regulating the cell-surface expression of PIP2;7 during abiotic stress conditions through protein-protein interaction and demonstrate an aquaporin regulatory mechanism involving TSPO.

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full‐length Arabidopsis trehalase is a plasma membrane‐bound protein, probably anchored to the membrane through a predicted N‐terminal membrane spanning domain. The full‐length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane‐bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae.

Pseudomonas fuscovaginae produces the lipodepsipeptides syringotoxin, fuscopeptin A and fuscopeptin B concurrently. These phytotoxins inhibit acidification of the external medium by fusicoccin-treated rice leaf sheath discs. When tested in vitro on H+-ATPase of rice shoot plasma membranes, syringotoxin and its structural analogue syringomycin, produced by P. syringae pv. syringae, displayed a double effect. At low concentrations they stimulated the ATPase activity of native right-side-out membrane vesicles in a detergent-like manner. At higher concentrations, however, this stimulation was reversed. With membranes treated with the detergent Brij 58, inhibition of ATPase activity was observed at low concentrations of the nonapeptides. The latter effect required the presence of an intact lactone ring formed by the nonapeptide head of these molecules. In contrast, fuscopeptins A and B inhibited enzyme activity regardless of the orientation of the vesicles. These observations were confirmed using plasma membranes from a yeast strain whose own H+-ATPase had been replaced by a single plant H+-ATPase isoform, PMA2, from Nicotiana plumbaginifolia. The kinetics of inhibition induced by the most active compound fuscopeptin B, showed a non-competitive pattern, with a Ki of about 1 microM. The combination of syringotoxin (or syringomycin) with the more hydrophobic fuscopeptins, in amounts with little or no effect, resulted in strong inhibition of the enzyme activity of rice membranes, suggesting a synergistic effect for the two types of toxins.

Enigmatic Translocator protein (TSPO) and cellular stress regulation

Translocator proteins (TSPOs) are conserved, ubiquitous membrane proteins identified initially as benzodiazepine-binding proteins in mammalian cells. Recent genetic and biochemical studies have challenged the accepted model that TSPOs are essential and required for steroidogenesis in animal cells. Instead, evidence from different kingdoms of life suggests that TSPOs are encoded by nonessential genes that are temporally upregulated in cells encountering conditions of oxidative stress, including inflammation and tissue injury. Here we discuss how TSPOs may be involved in complex homeostasis signaling mechanisms. We suggest that the main physiological role of TSPOs may be to modulate oxidative stress, irrespective of the cell type or subcellular localization, in part through the subtle regulation of tetrapyrrole metabolism.