Team
Team manager: Ieva Raffaele
Presentation
The team “Membrane Biogenesis and Protein Transport” addresses the fundamental processes at the basis of biogenesis and maintenance of cellular membranes, focusing on those encasing bacteria and eukaryotic cell organelles of bacterial origin, such as mitochondria.
We are particularly interested in the molecular mechanisms coordinating the multitude of processes that assemble distinct membrane building blocks. We focus on the recognition of protein targeting signals by protein transport machineries (translocases) and how the cooperative coupling of multiple translocases responds to signalling and environmental cues, fine-tuning the biogenesis of membrane bound compartments.
Towards these objectives, we combine omics and molecular approaches, including genome-wide screens, interactomic approaches, single particle cryoEM structural determination, gene/protein functional characterization in vivo and in vitro.
Our future orientations include the identification of inhibitory compounds that can function as new antimicrobials or as drugs against mitochondriopathies.
Project 1
Gram-negative bacteria represent a major public health concern due to their high resistance to antibiotics resulting in millions of human deaths world-wide each year. The outer membrane (OM) of Gram-negative bacteria plays a key role in virulence as it protects cells from physical and chemical stressors including several classes of antibiotics. Thus, the OM represents a priority target for the development of new antimicrobials.
Lipids and proteins contribute to the robustness of the OM. Integral outer membrane proteins (OMPs) play crucial roles in OM biogenesis and function, as they catalize protein and lipid assembly processes as well as the exchange of metabolites with the cell exterior.
A major objective of our lab is to elucidate the cellular mechanisms that coordinate efficient OM biogenesis, and how the OM undergos major remodeling events, for instance during cell division, while preserving its integrity. To this end, we study how the essential process of OMP assembly by the beta-barrel assembly machinery (BAM) is coordinated with other biogenesis and remodeling processes in the bacterial envelope. We have established a quantitative interactomic approach uncovering a dense network of interactions of BAM with other membrane assembly machineries, different types of envelope stress sensors and numerous uncharacterized proteins. The latter group includes two proteins that we have recently studied: DolP, an assembly factor of BamA, and LptM, a novel component of the lipopolysaccharide translocon (Ranava et al., 2021; Yang et al 2023, Chen et al., in preparation). Our current and future projects explore how these and other BAM interactors function at the molecular level.
Project 2
Mitochondria are organelles of bacterial origin that play central roles in eukaryotic cell metabolism, bioenergetics and signaling pathways. These functions are accomplished by a network of specialized multi-subunit complexes. Among these, the electron transport chain (ETC) complexes and the ATP synthase in mitochondrial cristae are well known for their coupled roles in oxidative phosphorylation, which produces the bulk of cellular ATP.
Mitochondria are made of approximately 1000 proteins, the majority of which are nuclear encoded and synthesized in the cell cytosol as precursor proteins. Many precursors possess cleavable amino-terminal presequence signals for mitochondrial targeting. These signals direct their import through the translocase of the outer membrane (TOM) and the presequence translocase of the inner membrane (TIM23). The presequence translocase-associated motor (PAM) contributes to energize preprotein import into the matrix. Upon import, many proteins such as the individual subunits of the ETC complexes undergo rapid assembly into their mature complexes, whereas unassembled components can generate off-pathway assembly intermediates at the detriment of mitochondrial proteostasis.
An important aim of our lab is to investigate how TOM and TIM23 orchestrate the step-wise process of preprotein import and assembly (Caumont-Sarcos et al., 2020; Kim et al., 2020; Turakhiya et al., 2016). In particular, we investigate how TIM23 guides imported proteins to their assembly lines, thereby streamlining the biogenesis of functional complexes, including these at the basis of oxidative phosphorylation. The coupling of mitochondrial protein import and assembly represents a potential regulatory hub that can rapidly modulate mitochondrial biogenesis in response to cellular signaling and metabolic cues.
– Yang Y., Chen H., Corey A., Morales V., Quentin Y., Froment C., Caumont-Sarcos A., Albenne C., Burlet-Schiltz O., Ranava D., Stansfeld P., Marcoux M., Ieva R. (2023) LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry. Nature Communications https://doi.org/10.1038/s41467-023-42007-w
– Ranava D#, Yang Y#, Orenday-Tapia L#, Rousset F, Turlan C, Morales V, Cui L, Moulin C, Froment C, Munoz G, Rech J, Marcoux J, Caumont-Sarcos A, Albenne C, Bikard D, and Ieva R. (2021) Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress. eLife https://doi.org/10.7554/eLife.67817
– Caumont-Sarcos A#, Moulin C#, Poinot L, Guiard B, van der Laan M, Ieva R. (2020) Transmembrane Coordination of Preprotein Recognition and Motor Coupling by the Mitochondrial Presequence Receptor Tim50. Cell Reports https://doi.org/10.1016/j.celrep.2020.02.031
– Lee S, Lee H, Yoo S, Ieva R, van der Laan M, von Heijne G, Kim H. (2019) The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop-transfer signals in the mitochondrial inner membrane. FEBS Letters https://doi.org/10.1002/1873-3468.13692
– Morales V., Orenday-Tapia L., Ieva R. (2024) Analysis of transmembrane beta-barrel proteins by native and semi-native polyacrylamide gel electrophoresis. Methods in Molecular Biology https://doi.org/10.1007/978-1-0716-3734-0_9
– Abuta’a YA, Caumont-Sarcos A, Albenne C, Ieva R. (2024) In Vivo Site-Directed and Time-Resolved Photocrosslinking of Envelope Proteins; Bacterial Secretion Systems: Methods and Protocols, Methods in Molecular Biology doi: https://doi.org/10.1007/978-1-0716-3445-5_20
– Moulin C, Caumont-Sarcos A, Ieva R. (2019) Mitochondrial presequence import: Multiple regulatory knobs fine-tune mitochondrial biogenesis and homeostasis. Biochim Biophys Acta Mol Cell Res doi: https://doi.org/10.1016/j.bbamcr.2019.02.012
Affiliation
