Biology subjects

Veith, K.

Publications and source records attributed to Veith, K..

6 recordsLinked to original sources

bioRxiv2025

Clathrin adaptors drive phase separation in endocytosis and trafficking

Liquid-liquid phase separation (LLPS) underlies the formation of biomolecular condensates that organize cellular processes, including endocytosis and membrane trafficking. Although LLPS has been implicated in clathrin-mediated pathways, the specific contribution of adaptor proteins to condensate formation and function remains unclear. Here we show that two yeast adaptors, Ent5 and Sla2, undergo LLPS in vitro and that this property correlates with their distinct roles in membrane trafficking and condensate recruitment in vivo. Ent5 condensation is driven by a dynamic helix within its disordered region that acts as a molecular "sticker." Deletion of this helix disrupts membrane-associated condensation and leads to altered cargo trafficking dynamics in vivo, with Ent5-dependent transport events becoming delayed. In contrast, Sla2 behaves both as a driver and as a client of LLPS, with its coiled-coil (CC) region mediating condensation. Together, these findings reveal that endocytic adaptors can promote condensate formation through distinct structural features, thereby coupling clathrin binding and membrane association through phase separation.

biophysics↗

bioRxiv2024

Subtleties in Clathrin Heavy Chain Binding Boxes provide selectivity among Adaptor Proteins of Budding Yeast

Clathrin, forming the triskelion network, orchestrates highly regulated cellular processes facilitating cargo internalization and trafficking in eukaryotes, with its N-terminal domain (NTD) pivotal for adaptor protein (AP) interactions. The NTD contains up to four AP-binding sites, and their roles in preferential occupancy by APs have not been addressed. Here, employing a combination of integrative biophysical and structural approaches together with in vivo functional experiments, we investigated the binding hierarchy and selectivity of adaptors for clathrin, aiming to understand the evolutionary conservation of redundant APs and their specialized roles in endocytosis and cellular trafficking mechanisms. We found that yeast epsin Ent5 displayed the highest affinity for clathrin, indicating its significant role in cellular trafficking processes. Epsins Ent1 and Ent2, which are crucial for endocytosis but described to have redundant functions, revealed distinct binding patterns; Ent1 demonstrated stronger interactions with clathrin than Ent2, explaining its functional divergence towards actin binding. Despite both having actin anchoring domains, since Ent1 is actually more stably recruited by clathrin, it would provide a better actin anchoring function. These results offer molecular insights into AP selectivity, suggesting they competitively bind clathrin while also targeting different clathrin sites.

molecular biology↗

bioRxiv2022

The Cryo-EM Structures of two Amphibian Antimicrobial Cross-β Amyloid Fibrils

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs) across kingdoms of life. Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-{beta} fibrils of mated {beta}-sheets at atomic resolution. Uperin 3.5 displayed substantial polymorphism with a protofilament of two mated {beta}-sheets. The determined structure was a polymorph showing a 3-blade symmetrical propeller of nine peptides per fibril layer including tight {beta}-sheet interfaces. This cross-{beta} cryo-EM structure complements the cross- fibril conformation previously determined by a crystal structure, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked {beta}-sheets allowing a rounded compactness of the fibril. The kinked {beta}-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. The amyloidal properties of antimicrobial peptides shed light on a mechanism of regulation of animicrobial activity involving self-assembly and fibril morphological variations. Moreover, the known endurance of amyloid structures can provide a template for the design of sturdy antimicrobials.

molecular biology↗

bioRxiv2021

Structural insights into a novel family of integral membrane siderophore reductases

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from P. aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a novel fold with two tightly-bound heme molecules. In combination with functional studies these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

biochemistry↗

bioRxiv2020

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites.

biophysics↗

bioRxiv2019

Crystal structures of the outer membrane transporter FoxA provide novel insights into TonB-mediated siderophore uptake and signalling

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.

molecular biology↗