Biology subjects

Sahi, C.

Publications and source records attributed to Sahi, C..

6 recordsLinked to original sources

bioRxiv2025

PAM18-3, a J-domain protein, maintains mitochondrial integrity and plant growth and development in Arabidopsis thaliana

Mitochondrial protein import is indispensable for organelle biogenesis and function, and is powered by the evolutionarily conserved presequence translocase-associated motor (PAM) complex. In Arabidopsis thaliana, three paralogs: PAM18-1, PAM18-2, and PAM18-3, encode J-domain proteins homologous to yeast PAM18, which stimulates the ATPase activity of mitochondrial HSP70 (mtHSP70) during protein translocation. Here, we identify PAM18-3 as the most highly and ubiquitously expressed paralog and demonstrate its critical role in mitochondrial function and plant development. Genetic disruption of PAM18-3 caused severe vegetative and reproductive defects, including reduced root length, smaller rosette size with fewer leaves, decreased plant height, shorter siliques, reduced seed set, and increased seed abortion. These phenotypes were fully rescued in complemented lines expressing PAM18-3. Ultrastructural analyses revealed profound mitochondrial abnormalities in mutants, whereas chloroplast architecture remained unaffected. Functional assays showed reduced mitochondrial membrane potential, and altered respiratory flux with a compensatory induction of the alternative oxidase (AOX) pathway. Transcript profiling revealed upregulation of AOX genes and multiple components of the mitochondrial TIM23 import apparatus and associated chaperones. Import assays demonstrated reduced mitochondrial accumulation of canonical TIM23 substrates, including IDH, ATP{beta}, and SHMT1, confirming a defect in matrix protein translocation. Consistently, pam18-3 mutants accumulated elevated reactive oxygen species (ROS) and exhibited strong induction of mitochondrial dysfunction stimulon (MDS) genes, including key transcription factors mediating retrograde signaling. Together, our findings establish PAM18-3 as a central component of the mitochondrial protein import machinery, supporting plant growth and development in Arabidopsis thaliana.

plant biology↗

bioRxiv2025

AtDjB3 regulates Hsc70-1-mediated expression of the heat shock genes and thermotolerance in Arabidopsis

Heat stress disrupts protein homeostasis, triggering the heat shock response (HSR) to maintain cellular proteostasis. A key aspect of this response is the release of heat shock factors (Hsfs) from Hsp70-mediated attenuation under heat-shock conditions, making Hsp70 a central regulator of HSR. However, the role of Hsp70 co-chaperones in this process remains largely unexplored in plants. Our study identifies AtDjB3, a heat-inducible class II J-domain protein (JDP), as a critical modulator of HSR. We present microscopy and cell fractionation-based evidence to demonstrate that the loss of AtDjB3 impairs Hsc70-1 recruitment to heat-induced protein aggregates, thereby maintaining HsfA1d bound to Hsc70-1 in the cytoplasm. This inhibited the expression of many heat-inducible genes, including HSP70s, HSP90, HSP18.2, FES1A, HSFA2, and HSFA7A. Consistent with this, AtDJB3 mutants displayed compromised thermotolerance, as evidenced by their inability to survive a prolonged heat stress of 37{degrees}C. Conversely, overexpression of AtDJB3 conferred enhanced thermotolerance, further supporting its positive regulatory role in the HSR. We propose that AtDjB3 not only contributes to the solubilization of heat-induced protein aggregates but also promotes Hsf activation by diverting Hsc70-1 to these aggregates, thereby releasing Hsfs to drive the transcriptional heat shock response.

plant biology↗

bioRxiv2024

Prefoldin 5 is a microtubule-associated protein that suppresses Tau-aggregation and neurotoxicity

Tauopathies represent a major class of neurodegenerative disorders associated with intracellular aggregates of the microtubule-associated protein Tau. To identify molecular modulators of Tau toxicity, we used a genetic screen to identify protein chaperones whose RNAi-mediated knockdown could modulate hTauV337M-induced eye-ommatidial degeneration in Drosophila. This screen identified the Prefoldins Pfdn5 and Pfdn6 as strong modifiers of hTauV337M cytotoxicity. Consistent with the known function of Pfdn as a cotranslational chaperone for tubulin, Pfdn5 mutants showed substantially reduced levels of tubulin monomer. However, additional microtubule-related functions were indicated by the robust unexpected association of Pfdn5 with axonal microtubules in vivo, as well as binding with stabilized microtubules in biochemical assays. Loss of Pfdn5 resulted in neuromuscular junctions (NMJ) defects similar to those previously described in hTau-expressing flies: namely, increased supernumerary boutons and fewer microtubule loops within mature presynaptic boutons. Significantly, synaptic phenotypes caused by hTauV337M overexpression were also strongly enhanced in a Pfdn5 mutant background. Consistent with a role in modulating Tau toxicity, not only did loss of Pfdn5 result in increased accumulations of Tau-aggregates in hTauV337M expressing neurons, but also neuronal overexpression of Prefoldin strikingly ameliorated age-dependent neurodegeneration and memory deficits induced by pathological hTau. Together, these and other observations described herein: (a) provide new insight into Prefoldin-microtubule interactions; (b) point to essential posttranslational roles for Pfdn5 in controlling Tau-toxicity in vivo; and (c) demonstrate that Pfdn5 overexpression is sufficient to restrict Tau-induced neurodegeneration.

neuroscience↗

bioRxiv2024

Inclusion of Hsp70 co-chaperone and RNA binding activities facilitates optimal function of spliceosomal disassembly factor Cwf23 in intron-rich Schizosaccharomyces pombe

Cwc23 is an essential J-domain protein that collaborates with the disassembly factor Ntr1 to facilitate spliceosomal disassembly. Although Cwc23 orthologs have been identified in spliceosomal extracts of many eukaryotes, their functionality in intron-rich eukaryotes remains largely unexplored. Here, we investigate the functionality of Cwf23, an ortholog of Cwc23 in Schizosaccharomyces pombe. Our study reveals that while the interaction between Cwf23 and SpNtr1 is conserved, it is not essential in S. pombe. Additionally, the RNA recognition motif (RRM) in Cwf23 is crucial for its function, as mutations in the RRM affect both growth and pre-mRNA splicing. Unlike its budding yeast counterpart, the J-domain of Cwf23 is indispensable, as J-domain mutants cannot support cell viability. These findings suggest a new paradigm in which the presence of a functional RRM and the essential nature of the J-domain underscore the increased requirements for an RNA-binding Hsp70 co-chaperone machinery in spliceosomal remodelling in "intron-rich" eukaryotes.

molecular biology↗

bioRxiv2024

Cytosolic Class I J-domain proteins aid mitochondrial protein import and influence homeostasis in Arabidopsis thaliana.

Mitochondrial homeostasis heavily relies on import of numerous nuclear encoded proteins. Multiple chaperone machineries operate at the cytosolic and mitochondrial side of the bilayer to ensure the efficacy of this process. While the mechanisms and components involved in translocation of polypeptides into the mitochondria are known in great detail, the cytosolic events of mitochondrial protein import are poorly understood in plants. This study explores the role of two cytosolic Class I J-domain proteins (JDPs), atDjA1 and atDjA2, in mitochondrial homeostasis in Arabidopsis thaliana. We show that atDjA1 and atDjA2 uniquely interacted with outer mitochondrial surface receptors, Tom9, Metaxin, and OM64, which guide the import of numerous mitochondrial precursors. Knockout of atDjA1 or atDjA2 impeded seed germination under multiple abiotic stress conditions, with atDjA1 mutant being more sensitive. Both the mutants demonstrated decreased respiratory rates and enhanced accumulation of reactive oxygen species (ROS), indicating compromised mitochondrial function. Further, knockout of atDjA1 resulted in a significant decline in the number of active mitochondria as well as import of MTS-GFP. While atDjA2 mutants did not show a reduction in mitochondrial number, both the mutants had an activated mitochondrial retrograde signaling (MRS) further underscoring compromised mitochondrial homeostasis in these mutants. Our findings suggest that evolutionarily conserved cytosolic Class I JDPs preserve mitochondrial homeostasis by targeting specific receptors on the outer mitochondrial surface, facilitating the import of cytosolically synthesized mitochondrial precursors in Arabidopsis thaliana.

plant biology↗

bioRxiv2020

Identification of subfunctionalized aggregate-remodeling J-domain proteins in plants

Hsp70s and J-domain proteins (JDPs) are among the most critical components of the cellular protein quality control machinery, playing crucial roles in preventing and solubilizing cytotoxic protein aggregates. Bacteria, yeast and plants additionally have large, multimeric Hsp100-class disaggregases which, allow the resolubilization of otherwise "dead-end" aggregates, including amyloids. JDPs interact with aggregated proteins and specify the aggregate remodeling activities of Hsp70s and Hsp100s. Plants have a complex network of cytosolic Hsp70s and JDPs, however the aggregate remodeling properties of plant JDPs are not well understood. Here we identify evolutionary-conserved Class II JDPs in the model plant Arabidopsis thaliana with distinct aggregate remodeling functionalities. We identify eight plant orthologs of the yeast protein, Sis1, the principal JDP responsible for directing the yeast chaperone machinery for remodeling protein aggregates. Expression patterns vary dramatically among the eight paralogous proteins under a variety of stress conditions, indicating their subfunctionalization to address distinct stressors. Consistent with a role in solubilizing cytotoxic protein aggregates, six of these plant JDPs associate with heat-induced protein aggregates in vivo as well as colocalize with plant Hsp101 to distinct heat-induced protein aggregate centers. Finally, we show that these six JDPs can differentially remodel multiple model protein aggregates in yeast confirming their involvement in aggregate resolubilization. These results demonstrate that compared to complex metazoans, plants have a robust network of JDPs involved in aggregate remodeling activities with the capacity to process a variety of protein aggregate conformers.

molecular biology↗