Biology subjects

Hegde, R. S.

Publications and source records attributed to Hegde, R. S..

11 recordsLinked to original sources

bioRxiv2026

Design principles of human membrane protein topology

We have curated and annotated the topologic determinants for all human membrane proteins made at the endoplasmic reticulum (ER). This census of 4,863 proteins allowed us to systematically analyze the physical properties of their 20,546 TMDs and flanking soluble regions. Single-pass proteins house the majority of large exoplasmic and cytosolic domains, whereas multipass proteins overwhelmingly contain short loops and tails. All classes of transmembrane domains (TMDs) have positively charged cytosolic flanks, but negatively charged exoplasmic flanks feature primarily on TMDs inserted by Oxa1-family insertases. The TMD-pair, a topologic unit of two TMDs with a short exoplasmic loop, is the dominant building block of multipass proteins. TMD-pairs accommodate high-hydrophilicity and charge-containing TMDs crucial for multipass protein functions. We interpret these context-dependent TMD features in light of current mechanistic models for membrane protein biogenesis and function. Our findings have implications for the evolution of membrane proteomes and for engineering new membrane proteins.

cell biology↗

bioRxiv2025

Mechanism of chaperone recruitment and retention on mitochondrial precursors

Nearly all mitochondrial proteins are imported into mitochondria from the cytosol. How nascent mitochondrial precursors acquire and sustain import-competence in the cytosol under normal and stress conditions is incompletely understood. Here, we show that under normal conditions, the Hsc70 and Hsp90 systems interact with and redundantly minimize precursor degradation. During acute import stress, Hsp90 buffers precursor degradation, preserving proteins in an import-competent state until stress resolution. Unexpectedly, buffering by Hsp90 relies critically on a mitochondrial targeting signal (MTS), the absence of which greatly decreases precursor-Hsp90 interaction. Site-specific photo-crosslinking and biochemical reconstitution showed how the MTS directly engages co-chaperones of Hsc70 (St13 and Stip1) and Hsp90 (p23 and Cdc37) to facilitate chaperone retention on the mature domain. Thus, the MTS has a previously unappreciated role in regulating chaperone dynamics on mitochondrial precursors to buffer their degradation and maintain import competence, functions that may facilitate restoration of mitochondrial homeostasis after acute import stress. Significance statementO_LIMitochondrial proteins encoded by the nuclear genome are synthesized in the cytosol before their subsequent import into mitochondria. The factors that recognize mitochondrial precursors in the cytosol to maintain their import-competence are incompletely defined. C_LIO_LIUsing a systematic site-specific photo-crosslinking strategy, the authors find that the mitochondrial targeting signal (MTS) is directly recognized by co-chaperones of Hsc70 and Hsp90. The co-chaperones facilitate recruitment, retention, and remodeling of these general chaperones on the nascent precursor protein. C_LIO_LIChaperone retention becomes particularly important during mitochondrial stress, when precursors must avoid degradation during a prolonged period in the cytosol. C_LI

cell biology↗

bioRxiv2024

Architecture of the UBR4 complex, a giant E4 ligase central to eukaryotic protein quality control

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Our cryo-EM analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3 MDa ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Structural data illustrate how UBR4 binds substrate and extends K48-specific ubiquitin chains. Importantly, efficient substrate targeting depends on both pre-ubiquitination and specific N-degrons, with KCMF1 acting as key substrate filter. Furthermore, we show that the architecture of the E4 megacomplex is conserved across eukaryotes but with species specific adaptations, allowing UBR4 to perform its precisely tuned quality-control function in diverse cellular environments.

biochemistry↗

bioRxiv2024

Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology1-4. This diverse protein family mediates the ionotropic signals triggered by major neurotransmitters and includes{gamma} -aminobutyric acid receptors (GABAARs) and acetylcholine receptors (nAChRs). Receptor function is fine-tuned by a myriad of endogenous and pharmacological modulators3. A functional pLGIC is built from five homologous, sometimes identical, subunits, each containing a {beta}-scaffold extracellular domain (ECD), a four-helix transmembrane domain (TMD) and intracellular loops of variable length. Although considerable progress has been made in understanding pLGICs in structural and functional terms, the molecular mechanisms that enable their assembly at the endoplasmic reticulum (ER)5 in a vast range of potential subunit configurations6 remain unknown. Here, we identified candidate pLGICs assembly factors selectively associated with an unassembled GABAAR subunit. Focusing on one of the candidates, we determined the cryo-EM structure of an assembly intermediate containing two 1 subunits of GABAAR each bound to an ER-resident membrane protein NACHO. The structure showed how NACHO shields the principal (+) transmembrane interface of 1 subunits containing an immature extracellular conformation. Crosslinking and structure-prediction revealed an adjacent surface on NACHO for {beta}2 subunit interactions to guide stepwise oligimerisation. Mutations of either subunit-interacting surface on NACHO also impaired the formation of homopentameric 7 nAChRs, pointing to a generic framework for pLGIC assembly. Our work provides the foundation for understanding the regulatory principles underlying pLGIC structural diversity.

biochemistry↗

bioRxiv2024

Convergence of orphan quality control pathways at a ubiquitin chain-elongating ligase

Unassembled and partially assembled subunits of multi-protein complexes have emerged as major quality control clients, particularly under conditions of imbalanced gene expression such as stress, aging, and aneuploidy. The factors and mechanisms that eliminate such orphan subunits to maintain protein homeostasis are incompletely defined. Here, we show that the UBR4-KCMF1 ubiquitin ligase complex is required for efficient degradation of multiple unrelated orphan subunits from the chaperonin, proteasome cap, proteasome core, and a protein targeting complex. Epistasis analysis in cells and reconstitution studies in vitro show that the UBR4-KCMF1 complex acts downstream of a priming ubiquitin ligase that first mono-ubiquitinates orphans. UBR4 recognizes both the orphan and its mono-ubiquitin and builds a K48-linked poly-ubiquitin degradation signal. The discovery of a convergence point for multiple quality control pathways may explain why aneuploid cells are especially sensitive to loss of UBR4 or KCMF1 and identifies a potential vulnerability across many cancers.

cell biology↗

bioRxiv2024

Soluble αβ-tubulins reversibly sequester TTC5 to regulate tubulin mRNA decay

Microtubules, built from heterodimers of - and {beta}-tubulins, control cell shape, mediate intracellular transport and power cell division. The concentration of {beta}-tubulins is tightly controlled through a post-transcriptional mechanism involving selective and regulated degradation of tubulin-encoding mRNAs. Degradation is initiated by TTC5, which recognizes tubulin-synthesizing ribosomes and recruits downstream effectors to trigger mRNA deadenylation. Here, we have investigated how cells regulate TTC5 activity. Biochemical and structural proteomic approaches reveal that under normal conditions, soluble {beta}-tubulins bind to and sequester TTC5, preventing it from engaging nascent tubulins at translating ribosomes. We identify the flexible C-terminal tail of TTC5 as a molecular switch, toggling between soluble {beta}-tubulin-bound and nascent tubulin-bound states. Loss of sequestration by soluble {beta}-tubulins constitutively activates TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.

cell biology↗

bioRxiv2023

Structural analysis of the dynamic ribosome-translocon complex

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-EM and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61s lateral gate, widening Sec61s central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.

biochemistry↗

bioRxiv2023

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation

SUMMARYMicrotubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits impacts polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER in turn engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.

molecular biology↗

bioRxiv2022

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent family of proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour promoting across a range of cancer types. To date, the molecular targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as a direct interactor and phosphatase substrate of both EYA4 and EYA1, with pY445 on PLK1 being the primary target site. EYA-mediated dephosphorylation of PLK1 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and the PLK1-activating proteins BORA and CEP192. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a novel phosphotyrosine signalling network governing PLK1 and mitosis. This work provides a mechanism of cell killing for EYA phosphatase inhibitors with important therapeutic implications.

cell biology↗

bioRxiv2020

A unified evolutionary origin for SecY and YidC

Cells use transporters to move protein across membranes, but the origins of the most ancient transporters are unknown. Here, we analyse the ubiquitous protein-conducting channel SecY. Features conserved by its two duplicated halves suggest that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecYs halves detect exceptional similarity with the only other ubiquitous protein transporter, YidC. Their shared fold comprises a three-helix bundle interrupted by a helical hairpin. In YidC this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY it is transmembrane and forms the substrate-binding lateral gate helices. In both, the three-helix bundle forms a protein-conducting hydrophilic groove, delimited by a conserved hydrophobic residue. We propose that SecY originated as a homodimeric YidC homolog. Many YidC homologs now use this interface to heterodimerise with a conserved partner. Unification of the two ubiquitous protein transporters would reconstruct a key step in the evolution of cells.

molecular biology↗

bioRxiv2020

The architecture of EMC reveals a path for membrane protein insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results show that EMCs cytosolic domain contains a large, moderately hydrophobic vestibule that binds a substrates transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMCs proposed chaperone function.

biochemistry↗