Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein-lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein-lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein-lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein-lipid interactions in plant cell biology.
- Klíčová slova
- Genetically encoded biosensors, lipid manipulation, membrane lipid imaging, microscopy, peripheral membrane proteins, protein–lipid interactions,
- MeSH
- buněčná membrána * metabolismus MeSH
- membránové lipidy metabolismus MeSH
- membránové proteiny metabolismus MeSH
- rostlinné proteiny * metabolismus MeSH
- rostliny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- membránové lipidy MeSH
- membránové proteiny MeSH
- rostlinné proteiny * MeSH
Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.
- MeSH
- buněčná membrána metabolismus MeSH
- endocytóza * MeSH
- klathrin * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- klathrin * MeSH
Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.
- Klíčová slova
- CLAVATA3-CLAVATA1 signaling, TPLATE complex, clathrin-mediated endocytosis, shoot apical meristem maintenance,
- MeSH
- Arabidopsis * genetika MeSH
- endocytóza MeSH
- meristém genetika MeSH
- proteiny huseníčku * genetika MeSH
- receptory buněčného povrchu metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostliny metabolismus MeSH
- signální transdukce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CLV1 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku * MeSH
- receptory buněčného povrchu MeSH
- TPLATE protein, Arabidopsis MeSH Prohlížeč
Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.
- MeSH
- buněčná membrána metabolismus MeSH
- endocytóza * MeSH
- klathrin * genetika metabolismus MeSH
- ubikvitin metabolismus MeSH
- ubikvitinace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- klathrin * MeSH
- ubikvitin MeSH
The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.
- MeSH
- cukerné fosfáty * metabolismus MeSH
- protein-serin-threoninkinasy genetika MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostliny metabolismus MeSH
- signální transdukce MeSH
- trehalosa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cukerné fosfáty * MeSH
- protein-serin-threoninkinasy MeSH
- proteiny huseníčku * MeSH
- trehalosa MeSH
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. However, the mechanistic contribution of the individual TPC subunits to plant CME remains 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. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified 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 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.
- MeSH
- adaptorové proteiny signální transdukční chemie genetika MeSH
- buněčná membrána metabolismus MeSH
- endocytóza * MeSH
- geneticky modifikované rostliny MeSH
- krystalografie rentgenová MeSH
- membránové proteiny chemie MeSH
- proteinové domény MeSH
- proteiny huseníčku MeSH
- proteiny vázající vápník chemie genetika MeSH
- rostlinné proteiny chemie genetika MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky MeSH
- tabák genetika MeSH
- transport proteinů MeSH
- vazba proteinů * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- membránové proteiny MeSH
- proteiny huseníčku MeSH
- proteiny vázající vápník MeSH
- rostlinné proteiny MeSH
- TPLATE protein, Arabidopsis MeSH Prohlížeč
