Biology subjects

Rofidal, V.

Publications and source records attributed to Rofidal, V..

8 recordsLinked to original sources

bioRxiv2026

ABCE1-dependent translational control links Fe-S cluster biogenesis to parasite growth and lipid homeostasis in Toxoplasma gondii

Toxoplasma gondii relies on tightly regulated protein synthesis to adapt to diverse host environments and to progress through its developmental stages. Here, we investigated the role of the ATP-binding cassette protein ABCE1, a broadly-conserved factor involved in ribosome recycling and translational control. Using a conditional knockdown approach, we demonstrate that depletion of TgABCE1 severely impairs parasite growth and disrupts global protein synthesis, confirming its essential role in maintaining translational capacity. TgABCE1 function depends on the incorporation of iron-sulfur (Fe-S) clusters, likely mediated by the cytosolic iron-sulfur assembly (CIA) pathway component HCF101. Depletion of TgABCE1 phenocopies the defects observed in TgHCF101-depleted parasites, supporting a functional link between these proteins. Notably, loss of TgABCE1 also disrupts lipid homeostasis, resulting in the accumulation of lipid droplets. Together, these findings uncover a critical link between translational regulation, Fe-S cluster biogenesis, and lipid homeostasis, highlighting the central role of proteostasis in parasite survival and development.

microbiology↗

bioRxiv2026

Two anchoring proteins control daughter apical complex assembly in Toxoplasma gondii

During Toxoplasma gondii division, the apical complex--comprising the conoid, apical polar ring (APR), and preconoidal rings--assembles with precise spatiotemporal coordination to form functional daughter buds. Despite their essential roles in invasion, motility, and division, the scaffolding proteins orchestrating this ordered assembly have remained largely unidentified. Here, we identify and characterize RCC1-2 and APR8 as essential factors directing distinct, sequential phases of daughter cell apical complex construction. Both proteins are recruited with precise spatial and temporal dynamics to the daughter buds, where they function as scaffolds rather than static structural components. APR8 transiently occupies the basal region of the APR specifically in early daughter cells. It is dispensable for conoid and PCR initiation, yet its loss causes APR collapse, abolishes SPMT anchoring, and eventually arrests conoid maturation. In contrast, RCC1-2 localizes beneath the APR basal layer and persists throughout daughter cell development, where it contributes to stabilizing the attachment of SPMTs to the APR. Notably, in situ cryo-electron tomography further reveals that the interspersed pillars bridging SPMTs ends to the APR fail to form properly in RCC1-2-depleted parasites. These findings map a hierarchical RCC1-2/APR8-dependent scaffolding process that advances our understanding of parasite replication.

microbiology↗

bioRxiv2025

Functional analysis of FW2.2-LIKE proteins in tomato: SlFWL5 regulates leaf size and morphology.

The CELL NUMBER REGULATOR/FW2.2-like (CNR/FWL) gene family comprises PLAC8 domain-containing membrane-associated proteins. The CNR/FWL gene family was named in reference to its founding member, the FW2.2 gene, which determines fruit size in tomato via a negative regulation on cell divisions. The functional characterization of PLAC8 domain-containing CNR/FWL proteins in plants is still very scarce, and only recently the molecular and cellular mode of action of FW2.2 was identified as regulating plasmodesmata-mediated cell-to-cell communication. In the present study, we provided a functional analysis of SlFWL genes in tomato, aiming at investigating any direct role in the control of organ growth. Based on a combination of molecular and cellular approaches, we selected three SlFWL proteins, namely SlFWL2, -4 and -5, which are localized at the plasma membrane. Gain- and loss-of-function transgenic plants were generated to explore their putative role as regulators of organ growth in tomato. This allowed us to shed light more specifically on the critical involvement of SlFWL5 in leaf development. We here showed that SlFWL5 is a plasmodesmata-localized protein that regulates leaf size and morphology via cell expansion, most probably through cell-to-cell communication. This original finding underscores further the importance of the CNR/FWL family members in growth regulation. Highlight: The FW2.2-LIKE protein SlFWL5 is localized at plasmodesmata and may have a role in cell-to-cell communication to regulate cell expansion during leaf development.

plant biology↗

bioRxiv2024

HCF101 is a novel component of the CIA cytosolic iron-sulfur synthesis pathway in the human pathogen Toxoplasma gondii

Several key cellular functions depend on proteins harboring an iron-sulfur (Fe-S) cofactor. As these Fe-S proteins localize to several subcellular compartments, they require a dedicated machinery for cofactor assembly. For instance, in plants and algae there are Fe-S cluster synthesis pathways localizing to the cytosol, but also present in the mitochondrion and in the chloroplast, two organelles of endosymbiotic origin. Toxoplasma gondii is a plastid-bearing parasitic protist responsible for a pathology affecting humans and other warm-blooded vertebrates. We have characterized the Toxoplasma homologue of HCF101, originally identified in plants as a protein transferring Fe-S clusters to photosystem I subunits in the chloroplast. Contrarily to plants, we have shown that HCF101 does not localize to the plastid in parasites, but instead is an important component of the cytosolic Fe-S assembly (CIA) pathway which is vital for Toxoplasma. While the CIA pathway is widely conserved in eukaryotes, it is the first time the involvement of HCF101 in this pan-eukaryotic machinery is established. Moreover, as this protein is essential for parasite viability and absent from its mammalian hosts, it constitutes a novel and promising potential drug target.

microbiology↗

bioRxiv2023

The FW2.2/CNR protein regulates cell-to-cell communication in tomato by modulating callose deposition at plasmodesmata

The FW2.2 gene is the founding member of the CELL NUMBER REGULATOR (CNR) gene family. More than 20 years ago, FW2.2 was the first cloned gene underlying a Quantitative Trait Locus (QTL) governing fruit size/weight in tomato. However, despite this discovery, the molecular mechanisms by which FW2.2 acts as a negative regulator of cell divisions during fruit growth remain undeciphered. In the present study, we confirm that FW2.2 is a transmembrane spanning protein, whose both N- and C-terminal ends are facing the apoplast. We unexpectedly found that FW2.2 is located at plasmodesmata (PD). FW2.2 participates in the spatiotemporal regulation of callose deposition at PD via an interaction with Callose Synthases, which suggests a regulatory role in cell-to-cell communication by modulating PD transport capacity and trafficking of signaling molecules during fruit development.

plant biology↗

bioRxiv2021

Root membrane ubiquitinome under short-term osmotic stress

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic mediated stress. Plants use the ubiquitin (Ub) proteasome system to modulate protein content, and a role for Ub in mediating endocytosis and trafficking plant plasma membrane proteins has recently emerged. In this study, we used the K-{varepsilon}-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no Ub motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment, but putatively in protein internalization as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub ligases, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

plant biology↗

bioRxiv2021

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis in Toxoplasma

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention. Author summaryToxoplasma gondii is a ubiquitous unicellular parasite that harbours two organelles of endosymbiotic origin: the mitochondrion, and a relict plastid named the apicoplast. Each one of these organelles contains its own machinery for synthesizing iron-sulfur clusters, which are important protein co- factors. In this study, we show that interfering with either the mitochondrial or the plastidic iron- sulfur cluster synthesizing machinery has a profound impact on parasite growth. However, while disrupting the plastidic pathway led to an irreversible loss of the organelle and subsequent death of the parasite, disrupting the mitochondrial pathway led to conversion of the parasites into a stress resistance form. We used a comparative quantitative proteomic analysis of the mutants, combined with experimental validation, to provide mechanistic clues into these different phenotypic outcomes. Although the consequences of disrupting each pathway were manifold, our data highlighted distinct changes at the metabolic level. For instance, the plastidic iron-sulfur cluster synthesis pathway is likely important for maintaining the lipid homeostasis of the parasites, while the mitochondrial pathway is clearly crucial for maintaining their respiratory capacity. Interestingly, we have discovered that other mutants severely impacted for mitochondrial function, in particular the respiratory chain, are able to survive and initiate conversion to the stress resistance form. This illustrates a different capacity for T. gondii to adapt for survival in response to distinct metabolic dysregulations.

microbiology↗

bioRxiv2019

Evidence for the involvement of protein lysine methylation in the response of sensitive and tolerant Arabidopsis species to cadmium stress

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analyzed the importance of protein lysine methylation for plants to cope with cadmium. We analyzed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs is regulated by cadmium. Also, we showed that nine out of 23 A. thaliana mutants interrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked-out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.

plant biology↗