Role of the retromer in seeds and seedling development in Arabidopsis thaliana

Chez les eucaryotes, le rétromère est un complexe protéique composé d’un sous complexe SNX (Sorting Nexin) et d’une sous unité VPS (Vacuolar Protein Sorting) également appelé « core » rétromère. Le rétromère a été décrit comme un complexe régulant le transport des protéines membranaires au niveau de l’endosome. Chez Arabidopsis thaliana, les travaux de notre équipe ont démontré que ce complexe est impliqué dans différents processus développementaux tels que le développement de l’embryon, la maturation des protéines de réserves de la graine et l’initiation des racines secondaires. Dans ce travail, nous avons caractérisé la fonction du rétromère dans le développement des graines et des jeunes plantules d’Arabidopsis thaliana. D’une part, nous avons montré que VPS29 est nécessaire à la mise en place des réserves lipidiques de la graine. Nous avons identifié un nouveau « cargo » du complexe rétromère ; LTP6 (Lipid Transfer Protein 6) dont la perte de fonction engendre des phénotypes liés au métabolisme lipidique similaires à ceux du mutant vps29. Compte tenu de la localisation de LTP6 au niveau d’une structure intracellulaire spongieuse caractéristique du réticulum endoplasmique, le site de synthèse des corps lipidiques, nous supposons que le rétromère participe à la biogenèse des réserves lipidiques via sa fonction dans le trafic de ce nouveau « cargo ». D’autre part, nous avons mis en évidence que le « core » rétromère indépendamment de la sous-unité SNX est impliqué dans la mobilisation des réserves lipidiques, une fonction indispensable pour le développement des jeunes plantules. Nous avons montré que VPS29 est nécessaire à la translocalisation de la triacylglycérol lipase SDP1 (Sugar-Dependent 1) du peroxysome aux corps lipidiques, le compartiment de stockage des réserves lipidiques. Ces résultats nous ont permis d’envisager que le « core » rétromère pourrait emprunter de nouvelles voies de trafics intracellulaires entre des compartiments autre que l’endosome.In eukaryotes, the retromer is a complex composed of the SNX (Sorting Nexin) subcomplex and the VPS (Vacuolar Protein Sorting) subcomplex also called the core retromer. To date, the retromer is described as a key regulator of proteins trafficking around endosomal compartment. In Arabidopsis thaliana, our group has previously demonstrated that this complex is involved in several developmental pathways, as embryo development, seed storage protein maturation and lateral root emergence. In this work, we characterised the function of the retromer in seeds and seedling development in Arabidopsis thaliana. Firstly, we found that VPS29 is required for the formation of seeds storage lipid. We identified a new cargo of this complex; Lipid Transfer Protein 6 (LTP6). LTP6 lost of function induces similar phenotype than vps29 linked to lipid metabolism. Based on LTP6 localisation on an intracellular structure characteristic of endoplasmic reticulum, the site of OBs formation, we supposed that the retromer may act on oil bodies biogenesis by its function on LTP6 trafficking. Secondly, we demonstrated that the core retromer have a SNX-independent function in lipid reserves breakdown, which is essential for seedling establishment. We showed that VPS29 is required for translocation of the triacylglycerol lipase SDP1 (Sugar-Dependent-1) from the peroxisome to oil bodies, the lipid storage compartment. Altogether, these results allowed us to propose new intracellular route trafficking for VPS sub-complex between compartments other than the endosome

Rôle du rétromère dans le développement des graines et la croissance des jeunes plantules chez Arabidopsis thaliana

Chez les eucaryotes, le rétromère est un complexe protéique composé d un sous complexe SNX (Sorting Nexin) et d une sous unité VPS (Vacuolar Protein Sorting) également appelé core rétromère. Le rétromère a été décrit comme un complexe régulant le transport des protéines membranaires au niveau de l endosome. Chez Arabidopsis thaliana, les travaux de notre équipe ont démontré que ce complexe est impliqué dans différents processus développementaux tels que le développement de l embryon, la maturation des protéines de réserves de la graine et l initiation des racines secondaires. Dans ce travail, nous avons caractérisé la fonction du rétromère dans le développement des graines et des jeunes plantules d Arabidopsis thaliana. D une part, nous avons montré que VPS29 est nécessaire à la mise en place des réserves lipidiques de la graine. Nous avons identifié un nouveau cargo du complexe rétromère ; LTP6 (Lipid Transfer Protein 6) dont la perte de fonction engendre des phénotypes liés au métabolisme lipidique similaires à ceux du mutant vps29. Compte tenu de la localisation de LTP6 au niveau d une structure intracellulaire spongieuse caractéristique du réticulum endoplasmique, le site de synthèse des corps lipidiques, nous supposons que le rétromère participe à la biogenèse des réserves lipidiques via sa fonction dans le trafic de ce nouveau cargo . D autre part, nous avons mis en évidence que le core rétromère indépendamment de la sous-unité SNX est impliqué dans la mobilisation des réserves lipidiques, une fonction indispensable pour le développement des jeunes plantules. Nous avons montré que VPS29 est nécessaire à la translocalisation de la triacylglycérol lipase SDP1 (Sugar-Dependent 1) du peroxysome aux corps lipidiques, le compartiment de stockage des réserves lipidiques. Ces résultats nous ont permis d envisager que le core rétromère pourrait emprunter de nouvelles voies de trafics intracellulaires entre des compartiments autre que l endosome.In eukaryotes, the retromer is a complex composed of the SNX (Sorting Nexin) subcomplex and the VPS (Vacuolar Protein Sorting) subcomplex also called the core retromer. To date, the retromer is described as a key regulator of proteins trafficking around endosomal compartment. In Arabidopsis thaliana, our group has previously demonstrated that this complex is involved in several developmental pathways, as embryo development, seed storage protein maturation and lateral root emergence. In this work, we characterised the function of the retromer in seeds and seedling development in Arabidopsis thaliana. Firstly, we found that VPS29 is required for the formation of seeds storage lipid. We identified a new cargo of this complex; Lipid Transfer Protein 6 (LTP6). LTP6 lost of function induces similar phenotype than vps29 linked to lipid metabolism. Based on LTP6 localisation on an intracellular structure characteristic of endoplasmic reticulum, the site of OBs formation, we supposed that the retromer may act on oil bodies biogenesis by its function on LTP6 trafficking. Secondly, we demonstrated that the core retromer have a SNX-independent function in lipid reserves breakdown, which is essential for seedling establishment. We showed that VPS29 is required for translocation of the triacylglycerol lipase SDP1 (Sugar-Dependent-1) from the peroxisome to oil bodies, the lipid storage compartment. Altogether, these results allowed us to propose new intracellular route trafficking for VPS sub-complex between compartments other than the endosome.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Peroxisome extensions deliver the Arabidopsis SDP1 lipase to oil bodies

Lipid droplets/oil bodies (OBs) are lipid-storage organelles that play a crucial role as an energy resource in a variety of eukaryotic cells. Lipid stores are mobilized in the case of food deprivation or high energy demands-for example, during certain developmental processes in animals and plants. OB degradation is achieved by lipases that hydrolyze triacylglycerols (TAGs) into free fatty acids and glycerol. In the model plant Arabidopsis thaliana, Sugar-Dependent 1 (SDP1) was identified as the major TAG lipase involved in lipid reserve mobilization during seedling establishment. Although the enzymatic activity of SDP1 is associated with the membrane of OBs, its targeting to the OB surface remains uncharacterized. Here we demonstrate that the core retromer, a complex involved in protein trafficking, participates in OB biogenesis, lipid store degradation, and SDP1 localization to OBs. We also report an as-yet-undescribed mechanism for lipase transport in eukaryotic cells, with SDP1 being first localized to the peroxisome membrane at early stages of seedling growth and then possibly moving to the OB surface through peroxisome tubulations. Finally, we show that the timely transfer of SDP1 to the OB membrane requires a functional core retromer. In addition to revealing previously unidentified functions of the retromer complex in plant cells, our work provides unanticipated evidence for the role of peroxisome dynamics in interorganelle communication and protein transport

Dictyostelium ACAP-A is an ArfGAP involved in cytokinesis, cell migration and actin cytoskeleton dynamics

ACAPs and ASAPs are Arf-GTPase-activating proteins with BAR, PH, GAP and ankyrin repeat domains and are known to regulate vesicular traffic and actin cytoskeleton dynamics in mammalian cells. The amoeba Dictyostelium has only two proteins with this domain organization, instead of the six in human, enabling a more precise functional analysis. Genetic invalidation of acapA resulted in multinucleated cells with cytokinesis defects. Mutant acapA(-) cells were hardly motile and their multicellular development was significantly delayed. In addition, formation of filopodial protrusions was deficient in these cells. Conversely, re-expression of ACAP-A-GFP resulted in numerous and long filopodia-like protrusions. Mutagenesis studies showed that the ACAP-A actin remodeling function was dependent on its ability to activate its substrate, the small GTPase ArfA. Likewise, the expression of a constitutively active ArfA•GTP mutant in wild-type cells led to a significant reduction in filopodia length. Together, our data support a role for ACAP-A in the control of the actin cytoskeleton organization and dynamics through an ArfA-dependent mechanism

Analyses of SORTING NEXINs Reveal Distinct Retromer-Subcomplex Functions in Development and Protein Sorting in Arabidopsis thaliana

International audienceSorting nexins (SNXs) are conserved eukaryotic proteins that associate with three types of vacuolar protein sorting (VPS) proteins to form the retromer complex. How SNXs act in this complex and whether they might work independently of the retromer remains elusive. Here, we show by genetic and cell imaging approaches that the Arabidopsis thaliana SNX1 protein recruits SNX2 at the endosomal membrane, a process required for SNX1-SNX2 dimer activity. We report that, in contrast with the mammalian retromer, SNXs are dispensable for membrane binding and function of the retromer complex. We also show that VPS retromer components can work with or independently of SNXs in the trafficking of seed storage proteins, which reveals distinct functions for subcomplexes of the plant retromer. Finally, we provide compelling evidence that the combined loss of function of SNXs and VPS29 leads to embryo or seedling lethality, underlining the essential role of these proteins in development

Mechanisms Governing the Endosomal Membrane Recruitment of the Core Retromer in Arabidopsis

International audienceThe retromer complex localizes to endosomal membranes and is involved in protein trafficking. In mammals, it is composed of a dimer of sorting nexins and of the core retromer consisting of vacuolar protein sorting (VPS)26, VPS29, and VPS35. Although homologs of these proteins have been identified in plants, how the plant retromer functions remains elusive. To better understand the role of VPS components in the assembly and function of the core retromer, we characterize here Arabidopsis vps26-null mutants. We show that impaired VPS26 function has a dramatic effect on VPS35 levels and causes severe phenotypic defects similar to those observed in vps29-null mutants. This implies that functions of plant VPS26, VPS29, and VPS35 are tightly linked. Then, by combining live-cell imaging with immunochemical and genetic approaches, we report that VPS35 alone is able to bind to endosomal membranes and plays an essential role in VPS26 and VPS29 membrane recruitment. We also show that the Arabidopsis Rab7 homolog RABG3f participates in the recruitment of the core retromer to the endosomal membrane by interacting with VPS35. Altogether our data provide original information on the molecular interactions that mediate assembly of the core retromer in plants