Biology subjects

Pereira, G. C.

Publications and source records attributed to Pereira, G. C..

6 recordsLinked to original sources

bioRxiv2022

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cells energy (ATP) supplies-particularly troublesome for energy demanding cells like neurons and myocytes. Unsurprisingly then, dysfunctional import is implicated in disease. This study explores the consequences of import failure in mammalian cells; wherein, blocking the import machinery has profound effects on mitochondrial ultra-structure and dynamics, but, surprisingly, does not impact import. The explanation is an astonishing response involving intercellular mitochondrial transfer via tunnelling nanotubes: for the import of healthy mitochondria and jettisoning of those with jammed import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial protein import failure. One-Sentence SummaryA mitochondrial import rescue mechanism involving intercellular mitochondrial transport through tunneling nanotubes (TNTs).

cell biology↗

bioRxiv2022

The NanoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import within Intact Mammalian Cells

Only a few proteins (13 in humans) are encoded by the mammalian mitochondrial genome. Therefore, the other mitochondrial resident proteins (>1000) must be recruited via specialised import pathways. Protein import is critical for mitochondrial biogenesis and bioenergetic function and health; loss of function has been implicated with a wide range of pathologies. Despite this, our understanding of the kinetic and dynamics of import is somewhat limited, particularly within mammalian cells. Here, we report an adaptation of an assay system, established previously to monitor mitochondrial import into isolated yeast mitochondria, to quantitatively monitor mitochondrial import inside mammalian cells. The reporting is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a recombinant precursor protein destined for import. Following import successively through the TOM complex of the outer membrane and the TIM23 complex of the inner membrane, the complementary fragments combine to form an active luciferase. The resultant luminescent signal provides a sensitive, accurate, free of noise and continuous measure of protein import, enabling mathematical model fitting to identify and understand the steps that make up import. This advance allows detailed mechanistic examination of the transport process in live cells. In addition, the assay will enable characterisation of the protein import when the machinery is challenged; for example, in situations associated with disease. Moreover, the assay is compatible with high throughput for large data set collection and kinetic modelling, as well as for drug screening and characterisation. Our set-up also has the potential to be adapted for the analysis of alternative transport systems and different cell types, and even for multicellular model organisms.

biochemistry↗

bioRxiv2021

Towards a molecular mechanism underlying mitochondrial protein import through the TOM-TIM23 complex

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences, and comes in two flavours: the TIM23SORT complex mediates inner mitochondrial membrane (IMM) protein insertion and the TIM23MOTOR complex delivers proteins to the matrix. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how {Delta}{psi} and ATP hydrolysis, the two main driving forces for import through the TIM23MOTOR complex, contribute to the import of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the OMM and initiation of IMM transport by the pre-sequence. The rates of these steps are influenced by PCP properties such as size and net charge, but not total amount of PCP imported - emphasising the importance of collecting rapid kinetic data, achieved here, to elucidate mechanistic detail. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.

biochemistry↗

bioRxiv2021

Maintenance of Complex I and respiratory super-complexes by NDUF-11 is essential for respiratory function, mitochondrial structure and health in C. elegans

Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing expression by RNAi allowed development to the adult stage, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, resulting in excessive detrimental ROS production. Meanwhile, electron cryo-tomography highlight aberrant cristae morphology and widening of the inter-membrane space and cristae junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development highlights the importance of complex I/ super-complex maintenance. Their perturbation by this, or other means, is likely to be the cause of metabolic stress and disease.

molecular biology↗

bioRxiv2019

A ‘proton ratchet’ for coupling the membrane potential to protein transport

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism by which PMF can aid transport, and enabling a proton ratchet, wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

biochemistry↗

bioRxiv2018

A High-Resolution Luminescent Assay For Rapid And Continuous Monitoring Of Protein Translocation Across Biological Membranes

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds - yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods.

biochemistry↗