Biology subjects

Degliesposti, G.

Publications and source records attributed to Degliesposti, G..

4 recordsLinked to original sources

bioRxiv2023

Elongator is a microtubule polymerase selective for poly-glutamylated tubulin

Elongator is a tRNA-modifying complex that regulates the fidelity of protein translation. Recently, a moonlighting function of Elongator has been identified in regulating polarization of the microtubule cytoskeleton during asymmetric cell division. Elongator induces symmetry breaking of the anaphase midzone by selectively stabilizing microtubules on one side of the spindle. This polarizes the segregation of signalling endosomes containing cell-fate determinants to only one daughter cell, thus contributing to cell fate determination. Here, we unravelled the molecular mechanism by which Elongator controls microtubule dynamics. Elongator binds simultaneously to the tip of microtubules and also to free GTP-tubulin heterodimers via their C-terminal tails. Elongator thereby locally increases tubulin concentration at microtubule ends, which stabilizes microtubules by increasing their growth speed and decreasing their catastrophe rate. We show that the Elp123 and Elp456 subcomplexes bind to microtubules and free tubulin heterodimers, respectively, and that these activities must be coupled for Elongator to stabilize microtubules. Surprisingly, we found that Elp456 has strong selectivity towards polyglutamylated tubulin dimers. Hence, microtubules assembled by Elongator become selectively enriched with polyglutamylated tubulin. Therefore, Elongator can rewrite the tubulin code of growing microtubules, placing it at the core of cytoskeletal dynamics and polarization during asymmetric cell division.

biochemistry↗

bioRxiv2020

A cryo-EM structure of metazoan TRAPPIII, the multisubunit complex that activates the GTPase Rab1

The TRAPP complexes are highly conserved nucleotide exchange factors, with TRAPPIII activating Rab1 and TRAPPII acting primarily on Rab11. The two complexes share a core of small subunits that affect nucleotide exchange, but are distinguished by additional large subunits that are essential for activity in vivo, and are mutated in a range of human disorders. Crystal structures of the core subunits have revealed the mechanism of Rab activation, but how and why the large subunits associate with the core remains unclear. We report here a cryo-EM structure of the entire TRAPPIII complex from Drosophila. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact bound Rab1, indicating how the arms could alter the Rab specificity of the core. A lower resolution structure of TRAPPII shows a similar architecture, and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

cell biology↗

bioRxiv2020

Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with HSP90, functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, snRNPs and PI3 kinase-like kinases (PIKK) such as TOR and SMG1. PIKK stabilisation depends on an additional complex of TELO2, TTI1 and TTI2 (TTT), whose structure and function are poorly understood. We have now determined the cryo-EM structure of the human R2TP-TTT complex that together with biochemical experiments reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

biochemistry↗

bioRxiv2018

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

biochemistry↗