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.

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.

Fission and fusion machineries converge at ER contact sites to regulate mitochondrial morphology.

The steady-state morphology of the mitochondrial network is maintained by a balance of constitutive fission and fusion reactions. Disruption of this steady-state morphology results in either a fragmented or elongated network, both of which are associated with altered metabolic states and disease. How the processes of fission and fusion are balanced by the cell is unclear. Here we show that mitochondrial fission and fusion are spatially coordinated at ER membrane contact sites (MCSs). Multiple measures indicate that the mitochondrial fusion machinery, Mitofusins, accumulate at ER MCSs where fusion occurs. Furthermore, fission and fusion machineries colocalize to form hotspots for membrane dynamics at ER MCSs that can persist through sequential events. Because these hotspots can undergo fission and fusion, they have the potential to quickly respond to metabolic cues. Indeed, we discover that ER MCSs define the interface between polarized and depolarized segments of mitochondria and can rescue the membrane potential of damaged mitochondria by ER-associated fusion.