Jelokhani-Niaraki, M. | Search bioRxiv

bioRxiv2024

AtTOC159 Receptors are Targeted to the Chloroplast Outer Membrane by a β-Signal and Galactolipid-Specific Transit Peptide-Like Sequence at the C-Terminus

Chloroplasts, the essential organelles responsible for photosynthesis, rely on the coordinated import of nuclear-encoded proteins for their biogenesis and function. TOC159 receptors are critical components of the translocon at the outer membrane of chloroplasts (TOC complex), responsible for recognizing and importing preproteins. However, the mechanisms by which TOC159 itself is targeted and integrated into the chloroplast outer envelope are only partially understood. In this study, we explore the structural aspects of the C-terminal membrane (M) domain of Arabidopsis thaliana TOC159, with a focus on its role in chloroplast outer envelope targeting and TOC complex integration. Structural predictions using AlphaFold and far-UV circular dichroism (CD) spectroscopy experiments suggested that the M domain forms an integral {beta}-barrel that fuses with the {beta}-barrel of TOC75, forming a hybrid channel at the chloroplast outer envelope. We identified a novel bi-partite targeting signal within the M domain, composed of a C-terminal {beta}-signal with the consensus sequence G-Q-{Phi}-[ST]-{Phi}-[RK]-X-[SN]-[ST] and a transit peptide (TP)-like sequence. Using fluorescence microscopy and mutational analyses, we demonstrated that the {beta}-signal is essential for chloroplast targeting, while the TP-like sequence enhances targeting ejiciency. Furthermore, CD spectroscopy and Langmuir-Blodgett trough experiments revealed that the TP-like sequence preferentially interacts with galactolipids unique to the chloroplast outer membrane, particularly sulfoquinovosyl diacylglycerol (SQDG), facilitating chloroplast outer envelope targeting specificity. Overall, our findings provide a deeper understanding of TOC159 receptor targeting mechanisms, TOC complex biogenesis and highlight the broader significance of lipid-mediated targeting in intracellular protein trajicking. These results pave the way for future studies on the role of TP-like sequences in chloroplast outer membrane protein targeting and other novel targeting pathways more generally.

plant biology↗

bioRxiv2018

The role of matrix-oriented salt-bridge on the proton transport in a multimeric uncoupling protein 2

Uncoupling proteins (UCPs) are members of the mitochondrial carrier family (MCF) that transport protons across the inner mitochondrial membrane, thereby uncoupling electron transport from ATP synthesis. The stoichiometry of UCPs, and the possibility of co-existence of this protein as mono-meric and associated forms in lipid membranes remain an intriguing open question. In the current study, the tertiary structure of UCP2 was analyzed both experimentally and through molecular dynamics (MD) simulations. After recombinant expression of UCP2 in the inner membrane of E. coli, the protein was directly extracted from the bacterial membranes with a non-denaturing detergent and purified both as a pure monomer and as a mixture of monomers, dimers and tetramers. Both protein preparations were re-constituted in egg yolk lipid vesicles. Gel electrophoresis, circular dichroism spectroscopy and fluorescence methods were used to characterize the structure and the proton transport function of protein. UCP2 showed unique stable tetrameric forms in lipid bilayers. MD simulations using membrane lipids and principal component analysis support the experimental results and provided new molecular insights into the nature of noncovalent interactions in oligomeric UCP2. MD simulations indicate that UCP2 tetramers are asymmetric dimers of dimers, in which the interactions between the monomers forming the dimer are stronger than the interactions between the dimers within the tetramer. It is also shown that UCP2 has a specific tendency to form functional tetramers in lipid bilayers, capable of proton transport. The asymmetric nature of the UCP2 tetramer could act as a scaffold for regulating the activity of the monomeric units through cooperative intercommunication between these subunits. Under similar experimental conditions, the structurally comparable ADP/ATP carrier protein did not form tetramers in vesicles, implying that spontaneous tetramerization cannot be generalized to all MCF members.\n\nSTATEMENT OF SIGNIFICANCESelf-assembly of membrane proteins plays a significant role in their biological function. In this article, both experimental and computational evidence are provided for spontaneous tetramerization of one of the mitochondrial uncoupling proteins (UCP2) in model lipid membranes. It is also shown that the tetrameric form of UCP2 is capable of proton transport, which leads to regulation of ATP synthesis in mitochondrion. Molecular dynamics simulations confirm the presence of asymmetric UCP2 tetramers as a potential scaffold for regulating the activity of the monomeric units through mutual intercommunication. The outcome of this study provides a solid ground for potential co-existence of monomeric and multimeric functional forms of UCPs that contributes to a deeper molecular insight into their structure and function.

biophysics↗