What makes PGC-1α extraordinary is not that it binds DNA directly—it does not—but that it operates as a flexible molecular scaffold, docking onto nuclear receptors and transcription factors to amplify their output by recruiting chromatin-remodeling machinery. The protein’s N-terminal activation domain, spanning the first 170 amino acids, recruits histone acetyltransferases such as p300 and SRC-1, enzymes that loosen chromatin by acetylating histone lysines and thereby making promoter regions accessible to the transcriptional apparatus. Embedded within this region are three leucine-rich motifs—L1, L2, and L3—that serve as molecular velcro for different classes of nuclear receptors. The L2 motif, conforming to the canonical LXXLL consensus, mediates high-affinity interactions with ligand-activated receptors including PPARα, PPARγ, ERα, thyroid hormone receptors, and glucocorticoid receptors, where the motif docks into a hydrophobic cleft formed by the receptor’s AF-2 helix upon agonist binding. In contrast, the L3 motif—an inverted LLKYL sequence—serves as the primary binding site for estrogen-related receptors, orphan nuclear receptors that lack identified endogenous ligands and instead rely constitutively on PGC-1α coactivation to drive mitochondrial gene expression in metabolically demanding tissues.

The structural basis for this promiscuity lies in PGC-1α’s designation as an intrinsically disordered protein. Unlike conventional enzymes with rigid binding pockets, PGC-1α exists as a dynamic conformational ensemble that shifts continuously in solution, adopting ordered structure only upon docking to a partner. AlphaFold2 predictions confirm this flexibility, assigning an average predicted local distance difference test score of just 52.75—well below the threshold indicating reliable structure—with confidence concentrated only in the C-terminal RNA recognition motif and cap-binding motif. This plasticity enables the protein to engage PPARγ in brown fat, HNF4α in liver, and MEF2 family members in muscle, tailoring its transcriptional output to the available partner landscape. Yet the same disorder that confers functional versatility also imposes a metabolic liability: ectopically expressed PGC-1α exhibits a half-life of approximately 30 minutes, degraded by default through the ubiquitin-independent 20S proteasome unless stabilized by the NADH-dependent gatekeeper NQO1.

Beyond canonical coactivation, the C-terminal region of PGC-1α harbors a second, less appreciated layer of regulation centered on RNA processing. Two arginine/serine-rich domains between amino acids 565 and 631, together with an RNA recognition motif spanning residues 677–710, connect the protein to the Mediator complex, the nuclear export receptor NXF1, and the cap-binding complex. Through interactions with CBP80 within the cap-binding complex, PGC-1α participates in quality control of nascent transcripts, facilitating release of RNA polymerase II from promoter-proximal pausing via recruitment of P-TEFb. Proteomic analyses indicate that over 80 percent of PGC-1α C-terminal protein-protein interactions depend on RNA, and this RNA-dependent assembly localizes the protein to membraneless chromatin condensates formed through liquid-liquid phase separation. The practical consequence is that PGC-1α does not merely activate transcription—it shepherds the resulting mRNAs through capping, splicing, and nuclear export, directly regulating the cytoplasmic availability of transcripts encoding mitochondrial proteins such as TFAM and cytochrome c oxidase subunits.

This structural and interactional plasticity is further tuned by an elaborate post-translational modification landscape. Phosphorylation by p38 MAPK at three sites within the negative regulatory domain increases protein half-life 2.5-fold and disrupts binding of the repressor p160 myb-binding protein, while AMPK phosphorylation at T177 and S538 primes PGC-1α for enhanced coactivation of GLUT4 and mitochondrial genes. Conversely, insulin-activated Akt2 phosphorylates S570, reducing promoter occupancy and suppressing gluconeogenic gene expression without globally inhibiting the protein. The interplay between activation and destruction is particularly elegant: p38 MAPK phosphorylation at T298 creates a priming site for GSK3β, which in turn generates a dual-phosphorylation degron recognized by the E3 ubiquitin ligase Fbw7, coupling transcriptional activation to subsequent proteasomal turnover. Lysine acetylation adds a further dimension—GCN5-mediated acetylation at 13 lysine residues redistributes PGC-1α to inactive nuclear compartments, whereas NAD+-dependent SIRT1 deacetylation restores coactivation of gluconeogenic genes in hepatocytes and fatty acid oxidation genes in skeletal muscle, directly transducing nutrient availability into transcriptional output. Additional modifications include arginine methylation by PRMT1 at R665, R667, and R669, which enhances ERRα coactivation; O-GlcNAcylation at S333, which stabilizes the protein by recruiting the deubiquitinase BAP1; and SUMOylation at K183, which represses activity by promoting association with the corepressor RIP140.

Given this central position in metabolic physiology, PGC-1α has become a tantalizing drug target for type 2 diabetes, obesity, neurodegeneration, and cancer. But the same intrinsic disorder that underpins its biological versatility has confounded rational drug design, as the protein lacks conventional binding pockets. The review catalogues the leading chemical modulators identified through high-throughput phenotypic screening. ZLN005, discovered from a library of 48,000 compounds, acts as an indirect activator by weakly uncoupling mitochondrial respiration, raising the AMP/ATP ratio and activating AMPK, which phosphorylates PGC-1α to drive a positive feedback loop involving MEF2C. In db/db diabetic mice, ZLN005 lowered fasting blood glucose and improved insulin sensitivity, though its efficacy across diverse pathological models—including ischemia-reperfusion injury, traumatic brain injury, and chronic kidney disease—has been tempered by a recent report that sustained administration following myocardial infarction worsened cardiac dysfunction, raising safety concerns. On the inhibitory side, SR18292 emerged from a screen of 350,000 compounds designed to enhance PGC-1α acetylation, selectively suppressing hepatic gluconeogenesis without altering mitochondrial gene expression. The compound redirects gluconeogenic precursors toward oxidative metabolism rather than lipogenesis, offering a mechanistically distinct approach to glycemic control.

The clinical stakes of this regulatory architecture are underscored by human genetics. The common Gly482Ser missense polymorphism in PPARGC1A is associated with increased type 2 diabetes risk across multiple populations and has been linked to nonalcoholic fatty liver disease and hypertrophic cardiomyopathy. In vitro studies indicate this variant displays reduced stability and diminished coactivator activity. Additional variants correlate with age of onset in Huntington’s disease, age of death in amyotrophic lateral sclerosis, and susceptibility to familial breast and colorectal cancers—reflecting PGC-1α’s dual role in supporting both tumor metabolic flexibility and p53-mediated growth arrest depending on interaction context.

The most provocative emerging insight concerns the protein’s role in cancer metabolism. PGC-1α interacts with wild-type p53 during early glucose starvation to promote cell cycle arrest and ROS clearance, yet mutant p53 variants bind PGC-1α with divergent affinities that determine whether tumor cells maintain metabolic flexibility for metastasis. In breast cancer patients carrying the R72 p53 polymorphism, the weakened interaction with PGC-1α leaves more coactivator available for ERRα-driven mitochondrial biogenesis, correlating with lower survival rates. Conversely, androgen receptor coactivation by PGC-1α promotes castration-resistant prostate cancer progression. This dual identity—tumor suppressor in some contexts, oncogenic enabler in others—reflects not an intrinsic property of the coactivator but the outcome of partner selection and cellular state, a distinction that any therapeutic strategy targeting PGC-1α must navigate with precision.

The review’s authors acknowledge that substantial gaps remain. The specific lysine residues targeted by ubiquitin ligases have not been conclusively mapped, conflicting models persist regarding which Fbw7 isoform drives degradation, and the molecular mechanisms governing PGC-1α nuclear trafficking remain unresolved despite correlative evidence linking exercise-activated kinases to nuclear accumulation. Tagging artifacts may underlie some discrepancies—GFP-tagged constructs exhibit nuclear distributions distinct from endogenous protein, and fluorescent tags are known to alter the localization of hundreds of proteins in a position-dependent manner.

As the field moves forward, the integration of structural disorder, isoform diversity, post-translational modification crosstalk, and RNA-mediated functions positions PGC-1α not as a simple on-off metabolic switch but as a signal-responsive regulatory hub whose output depends on the temporal and spatial convergence of dozens of inputs. The identification of small-molecule modulators, however imperfect their mechanisms, provides proof of concept that this notoriously disordered protein can be pharmacologically engaged. Whether future agents can achieve the tissue selectivity and temporal control that the biology demands—activating thermogenesis in adipose tissue while sparing the liver, or suppressing gluconeogenesis without compromising mitochondrial capacity in muscle—will determine whether the two decades of molecular dissection culminate in clinically useful therapeutics for the metabolic disorders that now burden hundreds of millions worldwide.

Mitochondria are double-membraned organelles that descended from free-living bacteria and still retain their own small genome, yet the overwhelming majority of the roughly thousand different proteins they need to function are encoded in the nucleus and synthesized on cytosolic ribosomes. Getting those proteins across two membranes, each with a distinct lipid composition and protein complement, is one of the defining logistical problems of eukaryotic cell biology. Mitochondria have solved it with a set of dedicated translocases, each specialized for a particular class of cargo, and the accuracy of the system matters enormously: mistargeted or unimported proteins can aggregate, clog membranes, or fail to deliver the metabolite transport that tissues such as heart, brain, and muscle depend on.

Among the most critical cargoes are the metabolite carriers of the SLC25 family, the largest solute transporter family in humans, which shuttle ADP and ATP, phosphate, fatty acids, and other essential metabolites across the otherwise impermeable inner membrane. Without these carriers, the organelle cannot exchange the chemical energy currency it produces with the rest of the cell, and cellular respiration grinds to a halt. These carriers are synthesized without cleavable presequences; instead, their six hydrophobic transmembrane segments must traverse the outer membrane through the TOM complex, cross the aqueous intermembrane space with the help of small Tim chaperones, and insert into the inner membrane through the TIM22 complex. In budding yeast, decades of work suggested that TOM and TIM22 act as separate stations along this pathway, connected only transiently by soluble Tim9–Tim10 chaperone complexes that ferry hydrophobic transmembrane segments through the space between the membranes. Yeast genetics and biochemistry had established the individual components of this route in considerable detail, which made the human system a natural point of comparison — and, as it turns out, a source of surprises.

The new study shows that human mitochondria operate differently. By expressing carrier substrates engineered for stable engagement of the import machinery, the researchers found that human TOM and TIM22 form a stable supercomplex detectable by blue native PAGE and pulldown assays from HEK293 cells. The supercomplex could be assembled in vitro using carrier substrates loaded onto Tim9–Tim10 chaperones, and its formation depended on the presence of an engaged substrate rather than occurring spontaneously in its absence, indicating that the carrier itself nucleates the junction between the two translocases. That substrate-dependence is functionally meaningful: it suggests the cell does not maintain a wasteful permanent bridge between membranes, but instead builds one on demand, precisely when a hydrophobic cargo molecule needs protection.

To visualize the transfer process, the team used cryo-electron microscopy on purified supercomplexes. They obtained maps of the substrate-engaged TOM complex and of the full TOM–TIM22 assembly, with structures deposited in the Protein Data Bank under accession codes 9WV1, 9WV2 and 9WV3 and maps in the EM Data Bank under EMD-66278 through EMD-66281. Cryo-EM has become the method of choice for interrogating membrane-embedded molecular machines of this size and fragility, because it images individual particles frozen in vitreous ice rather than requiring crystallization, and it has repeatedly revealed transient assemblies — like this one — that would never survive the rigors of crystallography. The structures reveal the carrier substrate threaded through the Tom40 channel with its transmembrane segments unpaired — unfolded and stretched out in single-helix form rather than packed as they are in the folded carrier — following a hydrophobic path along the inside of the channel wall. Notably, the substrate does not exit the bottom of the Tom40 pore as might have been expected. Instead, the transmembrane segments leave through an unexpected lateral groove on the side of the channel, emerging directly into the intermembrane space outside the main channel axis.

The researchers used photocrosslinking to map precisely where the substrate sits along its journey. By placing photoreactive probes at defined positions in the carrier transmembrane segments and the surrounding machinery, they showed that the carrier helices contact conserved hydrophobic residues lining the Tom40 channel, then transfer to the membrane-bound small Tim subunits, which in the human system include components that remain tethered to the inner membrane. These membrane-anchored small Tim proteins provide the entry site for the TIM22 complex, positioning the incoming hydrophobic segments for insertion. On the inner membrane side, a groove exposed on the membrane surface of TIM22 serves as the exit route through which carrier transmembrane segments leave the translocase and partition into the lipid bilayer. Together, the pathway forms a continuous, shielded route that keeps the aggregation-prone hydrophobic segments in a proteinaceous environment from the moment they leave the cytosol until they are embedded in the inner membrane.

That shielding principle is worth emphasizing, because it recurs throughout mitochondrial biogenesis. Hydrophobic transmembrane helices exposed to the aqueous intermembrane space would be prone to aggregation and misfolding, and the cell deploys chaperones at every vulnerable juncture — cytosolic factors on the way to the organelle, small Tim complexes in the space between membranes, and the translocase channels themselves as conduits. The supercomplex architecture takes this logic to its conclusion by eliminating any interval in which the cargo is not held by some component of the import machinery.

A central question was how the two membrane-embedded translocases physically connect across the intermembrane space. The structures show that Tim29, a subunit of the human TIM22 complex known to be required for carrier transport, participates in mediating supercomplex formation, along with Tim10b and the AGK subunit — the acylglycerol kinase that is mutated in Sengers syndrome and was previously identified as a TIM22 component. The authors tested the functional relevance of these connections by generating knockout and complemented cell lines; disruption of the relevant regions in AGK impaired carrier import, while the presequence-dependent import pathway, tested with the matrix protein SOD2, was unaffected, indicating that the TOM–TIM22 coupling is specific to the carrier route rather than a general collapse of mitochondrial import. This kind of pathway specificity is a hallmark of well-regulated cellular infrastructure: one branch of the import system can be compromised while parallel routes continue to operate, at least initially, a feature that helps explain the tissue-specific patterns of disease seen when individual components are lost.

To trap the supercomplex in a state suitable for imaging, the team also designed a fusion substrate in which Tim22 itself was linked to transmembrane helices 5 and 6 of the carrier GGC1 and a superfolder GFP tag, allowing the assembled complex to be purified through tandem affinity tags and examined by single-particle cryo-EM. This design let the researchers model how the carrier segments occupy the TOM channel while the receiving TIM22 complex stands ready at the inner membrane, and the two sfGFP densities at the TOM entrance provided a consistent handle for alignment during data processing. The strategy reflects a common and often decisive tactic in modern structural biology: when a transient assembly is too short-lived to purify intact, investigators engineer a tether that locks the interaction long enough to image it, then interpret the captured state in light of independent biochemical evidence.

The comparison with yeast is striking. In yeast Tom40, the equivalent exit path is blocked, and the extended C-terminal segment of the yeast channel forces substrates to follow a different route, consistent with the notion that in yeast the two complexes function separately. The human structures therefore suggest convergent but distinct solutions to the same problem: keeping hydrophobic carrier segments soluble as they cross the intermembrane space. The authors propose a sequential insertion model in which carrier transmembrane segments move one at a time from the TOM lateral groove onto the small Tim chaperones and then into the TIM22 exit groove, in a handoff that is tightly coordinated because the two translocases are held in a fixed spatial relationship within the supercomplex. A sequential handoff of this kind also provides a built-in quality-control opportunity: a segment that fails to transfer cleanly can be recognized and the process halted before a partially inserted, potentially harmful carrier accumulates in the inner membrane.

The work also intersects with recent findings on other cargo. A related study reported that the kinase PINK1, central to Parkin-mediated mitophagy and Parkinson's disease biology, is imported through human TOM complexes organized in arrays with the channel VDAC. The new carrier-pathway structures show a distinct substrate conformation and a distinct exit route compared with the PINK1-engaged TOM complex, underscoring that a single outer-membrane channel can accommodate multiple translocation modes with different substrate paths through the same pore. Taken together, these results suggest that Tom40 should be viewed not as a uniform pore but as a versatile platform whose behavior depends on the client it engages and the partner complexes it recruits.

The study has limitations inherent to its approach. The structures were obtained with engineered substrates and, in some cases, fusion proteins designed to stabilize the assembly, so the conformations captured may not represent every transient state that occurs during import in living mitochondria. The supercomplex was detected and enriched in the presence of substrate overexpression or crosslinking reagents such as glutaraldehyde, leaving open how dynamic the TOM–TIM22 association is under native conditions and what fraction of the cellular translocase population participates in supercomplexes at any given time. The resolution of the maps varies locally, and the unstructured or mobile regions of the substrate could not be fully modeled, which the authors acknowledge by marking unmodeled densities in their analyses. These caveats are typical of snapshot structural biology, and resolving them will likely require complementary approaches such as live-cell imaging of tagged import components, quantitative measurements of supercomplex abundance in unmanipulated mitochondria, and trapping of intermediate states along the handoff pathway.

Nevertheless, the implications are broad. Mutations in carrier import machinery components, including AGK, are linked to severe human mitochondrial diseases, and defects in SLC25 carrier biogenesis compromise energy metabolism in every tissue. Sengers syndrome, for example, combines congenital cataracts, skeletal myopathy, and cardiomyopathy, a pattern consistent with the energy-intensive tissues most dependent on carrier-mediated metabolite exchange.

In the evolving landscape of regenerative medicine, mitochondrial transplantation emerges as a startlingly promising approach. This novel concept involves isolating intact, functional mitochondria and delivering them into cells experiencing mitochondrial insufficiency. Unlike gene or stem cell therapies, mitochondrial transplantation aims to rapidly reconstitute bioenergetics without genetically altering the host cell. However, despite encouraging preliminary findings in animal and cellular models, a fundamental understanding of how transplanted mitochondria interface with recipient cells remains elusive. Do these organelles penetrate the cellular membrane and integrate functionally? If so, through which cellular uptake mechanisms? And critically, can they sustain their bioenergetic roles once internalized?

A landmark study recently addressed these pivotal questions with unprecedented rigor. Led by Associate Professor Kosuke Kusamori at Tokyo University of Science, the research employed mesenchymal stromal cells (MSCs)—a cell type renowned for regenerative potential—as recipients for isolated mitochondria. By amalgamating advanced imaging modalities—including fluorescence microscopy, confocal imaging, flow cytometry, and electron microscopy—with comprehensive biochemical assays, the investigators mapped the trajectory and function of exogenous mitochondria within MSCs. This multifaceted approach allowed precise visualization and quantification of mitochondrial uptake, as well as functional assessments post internalization.

Initial experiments focused on the isolation of mitochondria while preserving their ultrastructure and functional capacity. The mitochondria extracted from MSCs demonstrated high purity, devoid of contaminants such as other cellular organelles or debris. Importantly, these isolated mitochondria retained robust ATP synthesis ability, indicating preserved bioenergetic integrity during the isolation process. Subsequent provision of these mitochondria to living MSCs and hepatocytes yielded remarkable enhancements in cellular health. Notable outcomes included increased cell proliferation rates and improved resistance to oxidative and chemical stressors, reflecting the mitochondria’s cytoprotective effect.

A central question was whether these beneficial effects required actual mitochondrial internalization by recipient cells. Time-course studies revealed a gradual, time-dependent uptake of mitochondria by MSCs, reaching significant intracellular accumulation over several hours. Electron microscopic analysis showcased mitochondria entrapped within membrane-bound vesicles inside the cytoplasm, confirming true internalization rather than superficial adherence. By employing specific pharmacological inhibitors to block clathrin-, caveolin-, CLIC/GEEC-, and actin-mediated endocytic pathways, the research uncovered that MSCs utilize multiple, overlapping mechanisms to engulf transplanted mitochondria. This multiplicity underscores a complex, multifaceted cellular uptake process differing from single-pathway endocytosis found in many other biological processes.

Functional assays corroborated that the internalized mitochondria remained bioenergetically active. MSCs receiving mitochondrial transplants demonstrated enhanced mitochondrial respiration, evaluated by oxygen consumption rate measurements, alongside increased ATP production. These effects displayed a dose-responsive relationship to mitochondrial concentration, emphasizing the therapeutic potential of modulating mitochondrial doses. The enhanced respiratory capacity, coupled with heightened proliferation and stress resistance, suggests that transplanted mitochondria can more than simply survive within host cells—they actively improve cellular metabolic competence.

The elucidation of these uptake pathways and their biological consequences lays a crucial foundation for advancing mitochondrial transplantation from bench to bedside. By harnessing natural endocytic routes, therapeutic protocols can be optimized to maximize mitochondrial delivery efficiency. Such precision could enable tailored approaches adapted to the unique endocytic profiles of different cell types or pathological states. Moreover, confirming that transplanted mitochondria retain functionality challenges previous skepticism regarding their intracellular fate and offers compelling evidence for mitochondrial therapy as a distinct biomedical field.

Currently, mitochondrial transplantation remains in preclinical research, with many regulatory, safety, and efficacy hurdles to overcome. Long-term studies are needed to assess the persistence and integration of transplanted mitochondria, potential immune responses, and effects on tissue homeostasis across diverse disease models. Ensuring the purity, consistency, and biological activity of isolated mitochondria is paramount for clinical translation. Nonetheless, the non-genetic nature of this approach could provide rapid interventions for acute mitochondrial failure, circumventing complexities linked to gene editing or stem cell integration.

The therapeutic applications of mitochondrial transplantation are vast and especially poignant in diseases marked by mitochondrial defects. Ischemia–reperfusion injury following heart attacks or strokes, neurodegenerative conditions such as Parkinson’s and Alzheimer’s diseases, and toxin-induced hepatic injury are all promising targets. Furthermore, mitochondrial therapy could revolutionize treatment paradigms in aging—a state intimately linked to mitochondrial decline—and other chronic conditions characterized by compromised cellular energetics.

Ultimately, this innovative research represents a leap forward in regenerative medicine and cellular bioengineering. Dr. Kusamori and his team’s work provides a rigorous scientific blueprint for developing mitochondrial therapy as a novel, precise, and powerful modality to restore cellular energy homeostasis. With continued investigation and refinement, mitochondrial transplantation holds the potential to transform clinical care for a spectrum of debilitating diseases, offering hope for treatments rooted in the restoration of life’s fundamental energy processes.

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Subject of Research: Cells
Article Title: Uptake mechanisms and functions of isolated mitochondria in mesenchymal stromal cells
News Publication Date: 29-Dec-2025
References: DOI: 10.1038/s41598-025-28494-5
Image Credits: Associate Professor Kosuke Kusamori, Tokyo University of Science, Japan
Keywords: Mitochondria, Mesenchymal stromal cells, Mitochondrial transplantation, Cellular bioenergetics, Endocytosis, Regenerative medicine, Oxidative phosphorylation, Cellular respiration, Cell proliferation, Mitochondrial therapy, Neurodegenerative diseases, Mitochondrial dysfunction