Biology subjects

Ganesan, I.

Publications and source records attributed to Ganesan, I..

3 recordsLinked to original sources

bioRxiv2026

A dynamic displacement mechanism drives protein import into mitochondria

Most mitochondrial proteins are produced in the cytosol and imported through the translocase of the outer mitochondrial membrane (TOM) to reach their final destination. Although this protein entry gate has been structurally characterized, it remains unclear how precursor proteins are handed off from the cytosolic receptor domains to the translocation pore. Here we show that the cytosolic domain of Tom22--traditionally viewed as the central TOM receptor--acts not as a structured scaffold but as a largely disordered, flexible segment that plays an active role in precursor transfer. Atomic-level structural techniques and in vivo experiments identified a conserved short linear motif that forms a transient !-helical element within this disordered domain. By binding to the canonical precursor protein binding sites of the receptors Tom20 and Tom70, this critical -helical segment acts as a precursor protein displacement element (PPDE). This competitive interaction facilitates the release of preproteins directly above the import pore, and thereby drives translocation across the outer mitochondrial membrane. These findings reveal that flexibility, rather than rigid structure, underlies the central transfer step of mitochondrial outermembrane protein translocation. Our results point to a versatile mechanism for ligand displacement in chaperone, receptor, and transport systems that must balance selective binding with efficient release.

cell biology↗

bioRxiv2023

Cell-penetrating peptides stimulate protein transport on the Twin-arginine translocation pathway: evidence for a membrane thinning and toroidal pore mechanism

The Tat pathway is essential for photosynthetic protein transport across plant thylakoid membranes and is also ubiquitous throughout prokaryotes and archaea. The Tat pathway is unique amongst protein translocation pathways as it specializes in transporting folded proteins driven by a proton motive force. Mechanistic details of the actual translocation step (s) of the pathway remain elusive. Here, we show that membrane thinning stimulates Tat transport and, conversely, membrane strengthening abolishes Tat transport. We draw parallels from the Tat transport mechanism to that of cell penetrating peptides and propose that the Tat pore could be toroidal in shape and lined by lipids, as in those formed by cell penetrating peptides. Significance StatementProtein translocation across membranes is a significant cellular activity in both prokaryotes and eukaryotes. The Tat pathway for protein translocation operates in bacteria, archaea, chloroplasts, and plant mitochondria. Its mechanism of action has been difficult to decipher, but recent evidence suggests it does not use a conical proteinaceous transport channel. Instead, it has been suggested to translocate proteins through lipid-lined toroidal pores set up by membrane thinning. This work supports that hypothesis by showing that membrane-thinning cell-penetrating peptides stimulate the Tat pathway in both chloroplasts and bacterial plasma membranes, and that membrane stabilization blocks the pathway. We believe this is the most direct evidence to date of the toroidal pore mechanism operating in the Tat pathway.

biochemistry↗

bioRxiv2017

Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, which has historically been the thought for the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that medium-sized tightly folded proteins such as the 22 kDa dihydrofolate reductase (DHFR) can be tolerated by TOC and TIC. Chimeric DHFR fused with RuBisCO small subunit transit peptide (tp22DHFR) was found to be imported into chloroplasts in complex with its stabilizing ligand, methotrexate (MTX), in a folded conformation. Following import, both mature tp22DHFR and MTX were found in the chloroplast stroma. A subsaturating concentration of MTX was used to exclude the possibility that MTX was stripped off tp22DHFR, independently imported into the chloroplasts, and reassociated with imported tp22DHFR. Independent MTX import was further excluded by use of fluorescein conjugated MTX (FMTX), which has very slow membrane transport rates relative to unconjugated MTX. The TOC/TIC pore size was determined by probing the translocons with particles of fixed diameter and found to be greater than 25.6 [A], large enough to support folded DHFR import. The pore size is also larger than those of the mitochondrial protein translocons that have a requirement for protein unfolding.\n\nSIGNIFICANCEThe chloroplast TOC and TIC translocons are responsible for the import of up to 95% of all chloroplast proteins and are therefore essential for plastid biogenesis and photosynthesis. However, the mechanisms of protein import into chloroplasts are not well understood. The TOC/TIC translocons have long been suggested to have a strong unfoldase activity relative to other comparable protein translocons. Here, we present data suggesting that this is not true, and that instead, they possess a relatively large pore size. This identifies TOC and TIC as rather unique protein translocons capable of transporting folded proteins across a double membrane barrier, which has important implications in the mechanisms of TOC/TIC function and biogenesis of photosynthetic proteins.\n\nClassification - Biochemistry

biochemistry↗