Biology subjects

Broutzakis, G.

Publications and source records attributed to Broutzakis, G..

3 recordsLinked to original sources

bioRxiv2025

Structural basis of apoptosis induction by the mitochondrial voltage dependent anion channel

The voltage-dependent anion channel (VDAC) is the main gateway for metabolites across the mitochondrial outer membrane1. In addition, VDAC oligomers have been associated with apoptosis at mitochondrial stress conditions2. However, the mechanistic and structural basis of VDACs capability to induce apoptosis pathways remains poorly understood. Here, we show with biochemical and structural methods that VDAC1 oligomerization triggers the dissociation of its N-terminal -helix (VDAC1-N) from the channel interior. We used advanced lipid nanodiscs as a tool to selectively trap VDAC1 in its canonical helix-inserted and helix-exposed state to facilitate a structural characterization of both conformations by cryo-electron microscopy. The results show that slight changes in the shape and dynamics of the VDAC1 {beta}-barrel suffice to release the N-terminal helix to the channel exterior. This conformational switch addresses the long-standing question how VDAC1 can regulate partner protein binding. To confirm this hypothesis, we performed interaction studies between VDAC1 in both conformational states and the anti-apoptotic partner protein BclxL using nuclear magnetic resonance spectroscopy and could detect binding only for the helix-exposed state. These insights enabled the X-ray structure determination of the BclxL-VDAC1-N complex at high resolution and provided atomistic details on the VDAC1-N binding mode at the BH3-groove in BclxL. Further biochemical assays showed that VDAC1-N promotes pore formation of the pro-apoptotic Bcl2 protein Bak by neutralizing BclxLs inhibitory activity. These findings suggest that stress-induced oligomerization of VDAC can trigger the exposure of its N-terminal -helix leading to the neutralization of anti-apoptotic Bcl2 proteins. This mode-of-action is reminiscent of BH3-only sensitizer Bcl2 proteins3 that are efficient inducers of Bax/Bak-mediated mitochondrial outer membrane permeabilization and ultimately apoptosis.

biophysics↗

bioRxiv2024

High-resolution structures of the UapA purine transporter reveal unprecedented aspects of the elevator-type transport mechanism.

UapA is an extensively studied elevator-type purine transporter from the model fungus Aspergillus nidulans. Determination of a 3.6[A] inward-facing crystal structure lacking the cytoplasmic N-and C-tails, molecular dynamics (MD), and functional studies have led to speculative models of its transport mechanism and determination of substrate specificity. Here, we report full-length cryo-EM structures of UapA in new inward-facing apo- and substrate-loaded conformations at 2.05-3.5 [A] in detergent and lipid nanodiscs. The structures reveal in an unprecedented level of detail the role of water molecules and lipids in substrate binding, specificity, dimerization, and activity, rationalizing accumulated functional data. Unexpectedly, the N-tail is structured and interacts with both the core and scaffold domains. This finding, combined with mutational and functional studies and MD, points out how N-tail interactions couple proper subcellular trafficking and transport activity by wrapping UapA in a conformation necessary for ER-exit and but also critical for elevator-type conformational changes associated with substrate translocation once UapA has integrated into the plasma membrane. Our study provides detailed insights into important aspects of the elevator-type transport mechanism and opens novel issues on how the evolution of extended cytosolic tails in eukaryotic transporters, apparently needed for subcellular trafficking, might have been integrated into the transport mechanism.

biophysics↗

bioRxiv2021

Interactions of cytosolic termini of the Jen1 monocarboxylate transporter are critical for trafficking, transport activity and endocytosis

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism and growth, as well as for survival in response to stress or cytotoxic drugs, in both prokaryotes and eukaryotes. In the yeast Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that has been used to dissect the molecular details underlying control of cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence supporting previously non-described roles of the cytosolic N- and C- termini in Jen1 biogenesis, PM stability and activity, through functional analyses of rationally designed truncations and chimeric constructs with UapA, a S. cerevisiae endocytosis-insensitive purine transporter from Aspergillus nidulans. Our results reveal a cryptic role of the N-terminal region and thus show that both cytosolic N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. In particular, we provide evidence that the N- and the C-cytosolic termini of Jen1 undergo transport-dependent dynamic intra-molecular interactions, which critically affect the mechanism of transport and turnover of Jen1. Our results support an emerging concept where the cytosolic tails of PM transporters control transporter expression and function, through flexible intra-molecular interactions with each other and the transmembrane core of the protein. This idea may be extended to other MFS members providing a deeper understanding of conserved, but also evolving, mechanisms underlying MFS transporter structure-function relationships.

microbiology↗