Biology subjects

Loureiro-Lopez, M.

Publications and source records attributed to Loureiro-Lopez, M..

3 recordsLinked to original sources

bioRxiv2022

Biochemically validated structural model of the 15-subunit IFT-B complex

Cilia are ubiquitous eukaryotic organelles important to cellular motility, signalling and sensory reception. Cilium formation requires intraflagellar transport for trafficking of structural and signalling components. The large MDa IFT-B complex constitutes the backbone of polymeric IFT trains that carry ciliary cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B sub-complexes leaving >50% of the IFT-B complex structurally uncharacterized. We have used recent advances in protein structure prediction as implemented in Alphafold to assemble a structural model for the 15-subunit IFT-B complex. The model was validated using crosslinking/MS data on reconstituted IFT-B complexes, X-ray scattering in solution and diffraction from crystals as well as site-directed mutagenesis and protein binding assays. The IFT-B structural model reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The >400[A] long IFT-B complex can roughly be divided into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our structural modelling and crosslinking/MS results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of two different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

biochemistry↗

bioRxiv2022

The IFT81-IFT74 complex enhances GTP hydrolysis to inactivate RabL2 during early steps of intraflagellar transport

Cilia are important organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small Rab-like GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery to regulate the initiation of IFT. We have mapped the interaction with RabL2 to residues 107-195 of CEP19, purified the RabL2-CEP19 complex to show that CEP19 is not a GTPase activator protein for RabL2. In contrast, a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis in RabL2 by 20-fold. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 is mapped to a 70 amino acid long coiled-coil region of IFT81/74. We present structural models for minimal IFT81/74-RabL2 complexes and demonstrate that the Chlamydomonas IFT81/74 complex enhances GTP hydrolysis of human RabL2 suggesting an ancient evolutionarily conserved function. Our results provide a mechanistic understanding of RabL2 function in the initiation step of IFT and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.

biochemistry↗

bioRxiv2019

Functional role of respiratory supercomplexes in mice: segmentation of the Qpool and SCAF1

Mitochondrial respiratory complexes assemble into different forms of supercomplexes (SC). In particular, SC III2+IV require the SCAF1 protein. However, the structural role of this factor in the formation of the respirasome (I+III2+IV) and the physiological role of SCs are controversial. Here, we study C57BL/6J mice harbouring either non-functional SCAF1, the full knock-out for SCAF1 or the wild-type version of the protein and found a growth and exercise phenotype due to the lack of functional SCAF1. By combining quantitative data-independent proteomics, high resolution 2D Blue Native Gel Electrophoresis and functional analysis of enriched respirasome fractions, we show that SCAF1 confers structural attachment between III2 and IV within the respirasome, increases NADH-dependent respiration and reduces ROS production. Furthermore, through the expression of AOX in cells and mice we confirm that CI-CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity. These data demonstrate that SC assembly, regulated by SCAF1, modulates the functionality of the electron transport chain.

molecular biology↗