Biology subjects

Van Leene, J.

Publications and source records attributed to Van Leene, J..

7 recordsLinked to original sources

bioRxiv2026

Integration of SYT1 Interactomics and Dual-Localization Proteomics Links ER-PM Contacts to Lignin Deposition

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

molecular biology↗

bioRxiv2026

The molecular architecture of the Arabidopsis callose synthase complex

ABSTRACTCallose synthase is responsible for the targeted deposition of the {beta}-1,3-glucan polymer, callose which underlines essential plant developmental processes, including cell division, pathogen defense or cell-cell communication. The architecture of the callose synthase complex (CALSC) as well as the molecular mechanisms of callose synthesis remain unknown. Here we report an integrative characterisation of the Arabidopsis CALS complex, with the most enriched subunits, CALS1, CALS2 and CALS3, forming its core. Structurally, CALSC assembles into a trimer, requiring the plant-specific Bag domain to mediate inter-subunit associations. The biological importance of CALSC assembly is highlighted by the simultaneous loss of CALS1 and CALS3, which abolishes plasmodesmal callose deposition and affects symplastic transport. Site-directed mutagenesis and molecular dynamics simulations depict the topology of the CALS1 active site in detail, including the components of the enzymatic reaction. We pinpoint the translocating tunnel through which the nascent glucan is delivered and mechanistically confirm the role of transmembrane helix 8 in regulating glucan export. Our work provides unprecedented insight into the molecular architecture of the CALSC and the distinct changes from maturation to activity at the plasma membrane, while showcasing the mechanisms involved in callose synthesis at the molecular level.

plant biology↗

bioRxiv2022

SnRK1 inhibits anthocyanin biosynthesis through both transcriptional regulation and direct phosphorylation and dissociation of the MYB/bHLH/TTG1 MBW complex.

Plants have evolved an extensive specialized secondary metabolism. The colorful flavonoid anthocyanins, for example, not only stimulate flower pollination and seed dispersal but also protect different tissues against high light, UV- and oxidative stress. Their biosynthesis is highly regulated by environmental and developmental cues and induced by high sucrose levels. Expression of the biosynthetic enzymes involved is controlled by a transcriptional MBW complex, comprising (R2R3) MYB- and bHLH-type transcription factors (TF) and the WD40 repeat protein TTG1. Anthocyanin biosynthesis is obviously useful but also carbon- and energy-intensive and non-vital. Consistently, the SnRK1 protein kinase, a metabolic sensor activated in carbon- and energy-depleting stress conditions, represses anthocyanin biosynthesis. Here we show that Arabidopsis SnRK1 represses MBW complex activity both at the transcriptional and post-translational level. In addition to repressing expression of the key transcription factor MYB75/PAP1, SnRK1 activity triggers MBW complex dissociation, associated with loss of target promoter binding, MYB75 protein degradation and nuclear export of TTG1. We also provide evidence for direct interaction with and phosphorylation of multiple MBW complex proteins. These results indicate that repression of expensive anthocyanin biosynthesis is an important strategy to save energy and redirect carbon flow to more essential processes for survival in metabolic stress conditions.

plant biology↗

bioRxiv2022

Phase separation-based visualization of protein-protein interactions and kinase activities in plants

Protein activities depend heavily on protein complex formation and dynamic post-translational modifications, such as phosphorylation. Their dynamic nature is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization and high-end microscopy. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to study protein-protein interactions (PPIs) and kinase activities in planta based on phase separation. This technology enabled easy detection of inducible, binary and ternary protein-protein interactions among cytoplasmic, nuclear and plasma membrane proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SnRK1 kinase activity, allowing us to visualize tissue-specific, dynamic SnRK1 activation upon energy deprivation in stable transgenic Arabidopsis plants. The applications of the SYMPL cloning toolbox lay the foundation for the exploration of PPIs, phosphorylation and other post-translational modifications with unprecedented ease and sensitivity.

plant biology↗

bioRxiv2022

Mapping the adaptor protein complex interaction network in Arabidopsis identifies P34 as a common stability regulator

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargos and coated vesicle accessory proteins. Since in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargos of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins, and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

plant biology↗

bioRxiv2022

The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1, and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.

genetics↗

bioRxiv2020

Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Whereas one EH domain binds negatively charged PI(4,5)P2 lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

plant biology↗