Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Kivanç Birsoy
Abstract
Life systems across scales are organized as nested structures, in which each unit is assembled from and supported by spatially and functionally distinguishable subunits. In higher organisms, this principle, known as compartmentalization, applies to functional divisions ranging from organs and tissues to cells and subcellular structures. Compartmentalization is essential for supporting the complex functions of biological systems since it enables biological systems to dedicate concentrated resources and apply targeted regulations to highly specialized tasks. In eukaryotic cells, such functional specialization is achieved through subcellular compartments known as organelles. Many organelles are bound by lipid membranes, which create dedicated subspaces with distinct chemical environments that enable incompatible reactions to proceed in parallel. Moreover, membrane-bound organelles also harbor hydrophobic phases and membrane potential, thus serving as platforms for biosynthetic pathways, energy production and signaling functions. Decades of research have unraveled many essential processes that govern the biogenesis and homeostasis of organelles. However, much remains unknown regarding how organelles dynamically adjust their composition and functional output in response to ever-changing environmental challenges. Moreover, there is an unmet need for robust functional genomics tools that enable unbiased identification of the genetic determinants of many organellar properties and homeostasis mechanisms. This thesis attempts to address such challenges by elucidating novel mechanisms for organellar metabolite sensing and proteome homeostasis, as well as building new tools for biochemical assay-coupled forward genetics to identify unrecognized players in organellar biogenesis and quality control. A major unanswered question regarding subcellular metabolic homeostasis is how organelles robustly maintain stable internal concentrations of key metabolites against the fluctuating availability of nutrients. Focusing on glutathione, a metabolite that plays a key role in antioxidation defense and metal metabolism, we uncovered an autoregulatory mechanism that maintains its levels in the mitochondrial matrix. Glutathione is imported into mitochondria via SLC25A39, a mitochondrial inner membrane protein with a remarkably short half-life. Surprisingly, we found that a reduction in mitochondrial glutathione levels significantly stabilized SLC25A39 and adaptively promoted its import into the mitochondria. Mechanistically, glutathione is sensed via a cysteine-rich protruding loop on SLC25A39 that extends towards the mitochondrial matrix. Under glutathione-replete conditions, this loop mediates the recognition and rapid degradation of SLC25A39 by a membrane-anchored protease AFG3L2. Glutathione depletion promotes the binding of an [2Fe-2S] cluster to the cysteine residues on this loop, preventing the engagement of the AFG3L2 protease and promoting the adaptive increase in glutathione import. Such a mechanism enables the maintenance of a proper balance between mitochondrial iron and glutathione and safeguards mitochondrial protein complexes against the toxicity of excessive iron. Inspired by the key role of rapid proteolysis in metabolic control, as exemplified by SLC25A39, we hypothesized that additional mechanisms may employ effectors with short half-lives to implement dynamic and localized metabolic responses. Thus, we initiated an endeavor to screen dozens of putative short-lived proteins, which led to the identification of ABHD2 as a metabolic enzyme with exceptionally low stability. A genome-wide CRISPR screen revealed ABHD2 as a substrate of the MARCHF6-dependent ERAD pathway and surprisingly identified a poorly studied protein TXNDC15 as an essential functional partner of MARCHF6. Mechanistically, TXNDC15 appears to exert its ERAD-related function via a non-catalytic mechanism, potentially promoting the processing and translocation of its substrate by recruiting ER-localized glycan processing pathways. This work highlights the versatility in the mechanisms of action among protein degradation machineries as well as their roles in safeguarding metabolic homeostasis. The key role of these recently-deorphanized proteins and activities in organellar metabolic control underscores the demand for novel genetic tools tailored to organellar phenotypes. While a rich array of biochemical assays is available to extract multifaceted physical, morphological and functional phenotypes of subcellular components, many assays are incompatible with canonical functional genomics screens due to the loss of nuclear DNA from intact cells during cell lysis and organelle isolation. To overcome this challenge, we developed a functional genomics workflow, which we termed Subcellular Components Assay via sgRNA reLocalization and Targeted Transcript Isolation, or SCARLATTI, that involved targeting sgRNAs to arbitrary subcellular locations and recovering sgRNA sequences for forward genetic screens. We tested this workflow in biological processes ranging from protein degradation and processing, post-translational modification to organelle assembly and quality control. Using this new workflow, we identified numerous regulators involved in compartmentalized metabolic homeostasis, organelle assembly and quality control. Overall, this work provides conceptual advances and mechanistic insights into the compartmentalized metabolic control, and expands our genetic toolkit for untangling the complex biology in the subcellular space.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Liu, Yuyang, "Decoding the Logic of Compartmentalized Cellular Metabolism" (2026). Student Theses and Dissertations. 861.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/861
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy