UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum.

Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.

High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.

Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin-binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1ß and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum.

Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs. Significance Statement: UFM1 is a ubiquitin-like protein that is selectively conjugated to the large (60S) subunit of ribosomes bound to the endoplasmic reticulum (ER), but the specific biological function of this modification is unclear. Here, we show that UFMylation facilitates proteasome-mediated degradation of arrest polypeptides (APs) which are generated following splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER. We propose that UFMylation weakens the tightly sealed ribosome-translocon junction, thereby allowing the cytosolic ubiquitin-proteasome and ribosome-associated quality control machineries to access ER-APs.

STING induces LUBAC-mediated synthesis of linear ubiquitin chains to stimulate innate immune signaling.

STING activation by cyclic dinucleotides in mammals induces interferon- and NFκB -related gene expression, and the lipidation of LC3B at Golgi membranes. While mechanisms of the interferon response are well understood, the mechanisms of NFκB activation mediated by STING remain unclear. We report that STING activation induces K63- and M1-linked/linear ubiquitin chain formation at LC3B-associated Golgi membranes. Loss of the LUBAC E3 ubiquitin ligase prevents formation of linear, but not K63-linked ubiquitin chains or STING activation and inhibits STING-induced NFκB and IRF3-mediated signaling in monocytic THP1 cells. The proton channel activity of STING is also important for both K63 and linear ubiquitin chain formation, and NFκB- and interferon-related gene expression. Thus, LUBAC synthesis of linear ubiquitin chains regulates STING-mediated innate immune signaling.

The role of SwrA, DegU and P(D3) in fla/che expression in B. subtilis.

In B. subtilis swarming and robust swimming motility require the positive trigger of SwrA on fla/che operon expression. Despite having an essential and specific activity, how SwrA executes this task has remained elusive thus far. We demonstrate here that SwrA acts at the main σ(A)-dependent fla/che promoter PA(fla/che) through DegU. Electrophoretic mobility shift assays (EMSA) reveal that SwrA forms a complex with the phosphorylated form of DegU (DegU~P) at PA(fla/che) while it is unable to do so with either unphosphorylated DegU or the DegU32(Hy) mutant protein. Motility assays show that a highly phosphorylated DegU is not detrimental for flagellar motility provided that SwrA is present; however, DegU~P represses PA(fla/che) in the absence of SwrA. Overall, our data support a model in which DegU~P is a dual regulator, acting either as a repressor when alone or as a positive regulator of PA(fla/che) when combined with SwrA. Finally, we demonstrate that the σ(D)-dependent PD3(fla/che) promoter plays an important role in motility, representing a contingent feedback loop necessary to maintain basal motility when swrA is switched to the non-functional swrA(-) status.