Biology subjects

Denic, V.

Publications and source records attributed to Denic, V..

7 recordsLinked to original sources

bioRxiv2026

Retrograde trafficking inhibitors allosterically trap Get3 to block tail-anchored protein biogenesis

Retro-1 and Retro-2 are structurally distinct small molecules that protect cells from diverse toxins and viruses by disrupting retrograde trafficking, yet their mechanism of action has remained elusive. We show that both compounds target Get3, the ATPase chaperone of the guided entry of tail-anchored proteins (GET) pathway, which mediates biogenesis of tail-anchored SNARE proteins required for retrograde transport to the ER membrane. Cryo-electron microscopy reveals that Retro compounds bind a cryptic pocket in Get3, allosterically stabilizing Get3 in a stalled complex with upstream pathway components. Our work uncovers the GET pathway as an unsuspected vulnerability in pathogen entry, provides clear routes toward compound optimization, and establishes stabilization of dynamic protein complexes as a therapeutic strategy.

biochemistry↗

bioRxiv2024

A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant, multi-domain GTPase. Post-translational steps essential for eEF1A biogenesis are carried out by bespoke chaperones but co-translational mechanisms tailored to eEF1A folding remain unexplored. Here, we find that the N-terminal, GTP-binding domain of eEF1A is prone to co-translational misfolding and using computational approaches, yeast genetics, and microscopy analysis, we identify the conserved yet uncharacterized yeast protein Ypl225w as a chaperone dedicated to solving this problem. Proteomics and biochemical reconstitution reveal that Ypl225ws interaction with ribosomal eEF1A nascent chains depends on additional binding of Ypl225w to the UBA domain of nascent polypeptide-associated complex (NAC). Lastly, we show by orthogonal chemical genetics that Ypl225w primes eEF1A nascent chains for their subsequent binding to GTP and release from Ypl225w. Our work establishes eEF1A as a model system for chaperone-dependent co-translational folding and unveils a novel mechanism for GTP-driven folding on the ribosome.

biochemistry↗

bioRxiv2021

The peroxisomal exportomer directly inhibits phosphoactivation of the pexophagy receptor Atg36 to suppress pexophagy in yeast

Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 co-receptor, support a model in which exportomer represses Atg36 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.

molecular biology↗

bioRxiv2020

The importomer is a peroxisomal membrane protein translocase

Three sites of membrane protein biogenesis (the endoplasmic reticulum, mitochondria and chloroplasts) receive unfolded substrates from organelle-specific protein targeting factors, then integrate them using separate translocation channels. Peroxisomes also receive membrane proteins from known targeting factors, but whether a separate translocase is needed for integration remains unknown. Here, using a novel genetic screening strategy, we reveal that the importomer-known for matrix protein import-integrates the peroxisomal membrane protein Pex14. In importomer mutants, Pex14 is arrested in a pre-integrated state on the peroxisome surface. To undergo integration, a Pex14 translocation signal binds the importomers substrate receptor Pex5 at a distinct site from matrix proteins. En bloc translocation of an engineered protein complex with Pex14s luminal region argues that integration occurs without substrate unfolding. Our work shows that the handling of membrane protein targeting and integration by discrete machineries is a fundamental principle shared by diverse membrane protein biogenesis pathways.

cell biology↗

bioRxiv2019

The AAA+ ATPase TorsinA polymerizes into hollow tubes with a helical periodicity of 8.5 subunits per turn

TorsinA is an ER-resident AAA+ ATPase, whose single residue deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is a highly unusual AAA+ ATPase in that it needs an external activator. Also, it appears not to thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of the still enigmatic function of it. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 [A] structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner cavity of [~]4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA+ ATPases. We discuss the implications of these observations for TorsinA function.

biochemistry↗

bioRxiv2017

A new CRISPR screening approach for identifying novel autophagy-related factors and cytoplasm-to-lysosome trafficking routes

Selective autophagy comprises cytoplasm-to-lysosome trafficking routes that transport cargos using double-membrane vesicles (autophagosomes). Cargos are detected by receptor proteins, which typically also bind to lipid-conjugated LC3 proteins on autophagosome membranes. We dissected lysosomal delivery of four SQSTM1-like receptors by genome-wide CRISPR screening looking for novel autophagy-related (ATG) factors and trafficking routes. We uncovered new mammalian ATG factors including TMEM41B, an endoplasmic reticulum membrane protein required for autophagosome membrane expansion and/or closure. Furthermore, we found that certain receptors remain robustly targeted to the lysosome even in the absence of ATG7 or other LC3 conjugation factors. Lastly, we identified a unique genetic fingerprint behind receptor flux in ATG7KO cells, which includes factors implicated in nucleating autophagosome formation and vesicle trafficking factors. Our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

cell biology↗

bioRxiv2017

A New Quantitative Cell Microscopy Approach Reveals Mechanistic Insights Into Clearance Of Membrane Substrates By The AAA Protein Msp1

Msp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1. Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence.

cell biology↗