Biology subjects

Inglis, A. J.

Publications and source records attributed to Inglis, A. J..

5 recordsLinked to original sources

bioRxiv2026

Structural evolution of the MTCH family of mitochondrial insertases

We demonstrate that MTCH2 is the defining member of a large family of mitochondrial outer membrane (OM) insertases. MTCH insertases are conserved across holozoa and have diverged from the solute carrier 25 transporters. The cryoelectron microscopy structure of the 33 kDa human MTCH2 revealed that evolution of its insertase activity required loss of a transmembrane helix, which created a lipid-accessible hydrophilic groove stabilized by its unique, structured C-terminus. Mutational analyses showed that MTCH insertase activity is attenuated, while experimental structures and reconstitution of hyperactive mutants demonstrated that the hydrophobicity, charge, and size of the residues that line its groove regulated MTCH function. Leveraging the MTCH2 structure, we identified the plant OM insertase, and proposed a universal mechanism for OM insertion across all kingdoms of life. TeaserStructure of human MTCH2 reveals conserved features necessary to maintain mitochondrial proteome integrity across eukaryotes.

cell biology↗

bioRxiv2023

Triaging of -helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology

Mitochondrial outer membrane -helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse substrates remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse -helical substrates reveals that these components are organized into distinct targeting pathways which act on substrates based on their topology. NAC is required for efficient targeting of polytopic proteins whereas signal-anchored proteins require TTC1, a novel cytosolic chaperone which physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

cell biology↗

bioRxiv2022

MTCH2 is a mitochondrial outer membrane protein insertase

In the mitochondrial outer membrane, tail-anchored (TA) proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPRi screens, we identify factors involved in mitochondrial TA biogenesis in human cells. We show that MTCH2, and its paralog MTCH1, are required for insertion of biophysically diverse mitochondrial TAs, but not outer membrane {beta}-barrel proteins. In a reconstituted system, purified MTCH2 is sufficient to mediate insertion into proteoliposomes. Functional and mutational studies reveal that MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for outer membrane protein biogenesis, controlling mislocalization of TAs into the endoplasmic reticulum and the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction. One-Sentence SummaryMTCH2 is both necessary and sufficient for insertion of diverse -helical proteins into the mitochondrial outer membrane, and is the defining member of a family of insertases that have co-opted the SLC25 transporter fold.

cell biology↗

bioRxiv2021

Coupled protein quality control during nonsense mediated mRNA decay

Translation of mRNAs containing premature termination codons (PTCs) can result in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance path-way responsible for detecting and degrading PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on an intact ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, and suggested a lack of dependence on the canonical ribosome-quality control (RQC) pathway. Finally, one of the strongest hits in our screens was the E3 ubiquitin ligase CNOT4, a member of the CCR4-NOT complex, which is involved in initiating mRNA degradation. We show that CNOT4 is involved in NMD coupled protein degradation, and its role depends on a functional RING ubiquitin ligase domain. Our results demonstrate the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a framework for identifying and characterizing factors involved in this process.

molecular biology↗

bioRxiv2019

Differential modes of orphan subunit recognition for the WRB/CAML complex

A large proportion of membrane proteins must be assembled into oligomeric complexes for function. How this process occurs is poorly understood, but it is clear that complex assembly must be tightly regulated to avoid accumulation of orphan subunits with potential cytotoxic effects. We interrogated assembly in mammalian cells using a model system of the WRB/CAML complex: an essential insertase for tail-anchored proteins in the endoplasmic reticulum (ER). Our data suggests that the stability of each subunit is differentially regulated. In WRBs absence, CAML folds incorrectly, causing aberrant exposure of a hydrophobic transmembrane domain to the cytosol. When present, WRB can post-translationally correct the topology of CAML both in vitro and in cells. In contrast, WRB can independently fold correctly, but is still degraded in the absence of CAML. We therefore propose at least two distinct regulatory pathways for the surveillance of orphan subunits during complex assembly in the mammalian ER.

molecular biology↗