Biology subjects

Kraut, D. A.

Publications and source records attributed to Kraut, D. A..

4 recordsLinked to original sources

bioRxiv2025

STI1 domain dynamically engages transient helices in disordered regions to drive self-association and phase separation of yeast ubiquilin Dsk2

Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and ubiquitinated substrates under azide-induced mitochondrial stress or extended growth conditions. However, the molecular mechanisms underlying how these shuttle proteins work are unknown. Here, we identify that the middle chaperone-binding STI1 domain is the main driver of Dsk2 self-association and phase separation in vitro. Using NMR spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically-disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces the propensity for Dsk2 to phase separate. In vivo, removal of the STI1 domain in Dsk2 has the opposite effect, resulting in an increase of proteasome-containing condensates due to an accumulation of polyubiquitinated substrates. Modeling of STI1-helix interactions reveals a binding mode that is reminiscent of interactions between chaperone STI1/DP2 domains and client proteins containing amphipathic or transmembrane helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes. HighlightsO_LIThe intrinsically disordered regions of Dsk2 harbor transient helices that regulate protein properties via interactions with the STI1 domain. C_LIO_LIThe STI1 domain is a significant driver of Dsk2 self-association and phase separation in vitro. C_LIO_LIDsk2 colocalizes with ubiquitinated substrates and proteasome in reconstituted condensates. C_LIO_LIAbsence of Dsk2 STI1 domain in stressed yeast cells promotes formation of proteasome condensates coupled with upregulation of polyubiquitinated substrates. C_LI

biochemistry↗

bioRxiv2024

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2s phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code. SignificanceCovalent attachment of polyubiquitin chains to eukaryotic proteins is a common protein quality control signal. Ubiquitination often marks proteins for degradation by the proteasome, but can also drive non-degradative outcomes. Proteins, including UBQLN2, that bind both polyubiquitin and the proteasome can either enhance or inhibit degradation. The ALS-related UBQLN2 is recruited to membraneless organelles, including stress granules, and undergoes phase separation in vitro, but the effects of phase separation on substrate fate are unknown. Herein we show that UBQLN2 phase separation is modulated by polyubiquitinated substrates in a linkage-dependent fashion. We show that two functional outcomes, degradation and deubiquitination, are differentially affected by phase separation. Our results suggest that phase separation of substrates and UBQLN2 could control protein fates.

biochemistry↗

bioRxiv2024

Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway

In eukaryotes, the ubiquitin-proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore-1 loops of the RPs Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N-terminus, remain unknown. We determined how the location of glycine-rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine-rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding. ImportanceThe proteasome processively unfolds and degrades target proteins in eukaryotes. However, some substrates are prematurely released, and the resulting partially degraded proteins can cause problems for cells and can be linked to neurodegenerative diseases. In this paper, we use a series of substrates that can stall the proteasome during degradation to probe the translocation pathway substrates must traverse during unfolding. We find that multiple points along the translocation pathway are impacted by these slippery substrates.

biochemistry↗

bioRxiv2020

Mode of Targeting to the Proteasome Determines GFP Fate

The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasomes ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.

biochemistry↗