|Titel:||Structural and biophysical studies of mid-coat endocytic adaptors||Sprache:||Englisch||Autor*in:||Lizarrondo Diaz de Cerio, Javier||GND-Schlagwörter:||Structural biologyGND
|Erscheinungsdatum:||2022-10||Tag der mündlichen Prüfung:||2022-12-16||Zusammenfassung:||
Clathrin-mediated endocytosis is a conserved process in eukaryotes essential for several functions such as nutrition and synaptic communication. During clathrin-mediated endocytosis, a large protein machinery selectively assembles in a region of the plasma membrane, creating clathrin-coated pits (CCPs). Within this complex and dynamic network protein-protein and protein-membrane interactions, endocytic midcoat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination in yeast. This work presents a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2. The building unit from this oligomer is a hetero-tetramer that binds PIP2 molecules at the ANTH-ENTH interfaces. Structure-derived mutations of residues present in the newly identified protein interfaces of this complex were studied by a wide-range of biophysical and structural techniques. Complex formation experiments delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. Importantly, the ANTH-ENTH cooperate to form larger assemblies to contribute to membrane remodelling, promoting the formation of CCPs. Furthermore, a time-resolved small-angle X-ray scattering (TR-SAXS) study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast sub-second timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex.
Beyond the membrane binding domains of mid coat adaptors, biophysical characterization of full length Sla2 and Ent1 from C. thermophilum was also performed. cryoEM Single Particle Analysis of Sla2 together with structural predictions reveals different oligomeric and conformational states which could be relevant for its interaction with the actin cytoskeleton, an essential component requiered for membrane internalization.
Furthermore, interaction of adaptors with clathrin is also studied here: the complex between Sla2 and clathrin light chain (CLC) is characterised in solution using SEC-SAXS and binding studies reveal a low affinity protein-protein interaction which affects the flexibility of clathrin. The crystal structure of the clathrin heavy chain N-Terminal domain (NTD) from C. thermophilum shows a high conservation from other eukaryotes, and binding studies of this domain with adaptors shed some light regarding the specificity of the binding of these adaptors to the clatrin coat.
Together, these findings provide a molecular understanding of how mid-coat adaptors contribute to the molecular puzzle assembled for building clathrin-coated vesicles.
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|
geprüft am 02.10.2023
geprüft am 02.10.2023