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Ribosomal Protein RPL26 Is the Principal Target of Ufmylation

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Ribosomal Protein RPL26 Is the Principal Target of Ufmylation Ribosomal protein RPL26 is the principal target of UFMylation Christopher P. Walczaka, Dara E. Letoa, Lichao Zhangb, Celeste Riepec, Ryan Y. Mullerc, Paul A. DaRosaa, Nicholas T. Ingoliac, Joshua E. Eliasb, and Ron R. Kopitoa,1 aDepartment of Biology, Stanford University, Stanford, CA 94305; bDepartment of Chemical and Systems Biology, Stanford University, Stanford, CA 94305; and cDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Edited by Brenda A. Schulman, Max Planck Institute of Biochemistry, and approved December 10, 2018 (received for review September 19, 2018) Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, observed in embryonic lethal mouse knockouts of Uba5, Ufl1,or ubiquitin-like protein modifier that is essential for embryonic devel- Ddrgk1 (8–10), and cardiomyopathy ensuing from heart-specific opment. Although loss-of-function mutations in UFM1 conjugation disruption of Ufl1 (25), is accompanied by elevated markers of are linked to endoplasmic reticulum (ER) stress, neither the biological ER stress. Despite this circumstantial evidence linking the UFMy- function nor the relevant cellular targets of this protein modifier are lation system to the ER, reported UFMylation targets expressed at known. Here, we show that a largely uncharacterized ribosomal endogenous levels are neither physically nor physiologically asso- protein, RPL26, is the principal target of UFM1 conjugation. RPL26 ciated with the ER. Unambiguous identification of covalent, phys- UFMylation and de-UFMylation is catalyzedbyenzymecomplexes iological UFM1 targets holds the key to elucidating the biological function of this essential, metazoan-specific protein modification. tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is Here we combine genetic disruption of UFMylation and de- highly enriched on ER membrane-bound ribosomes and polysomes. UFMylation with affinity capture LC-MS/MS to show that the Biochemical analysis and structural modeling establish that UFMylated conserved ribosomal protein RPL26 (uL24) is the principal tar- RPL26 and the UFMylation machinery are in close proximity to the get of UFMylation in human cells. Our data show that ribosome- SEC61 translocon, suggesting that this modification plays a direct role bound RPL26 is covalently UFMylated and de-UFMylated by in cotranslational protein translocation into the ER. These data suggest enzymes tethered to the ER membrane. Inhibition of RPL26 that UFMylation is a ribosomal modification specialized to facilitate UFMylation or de-UFMylation leads to impaired ER protein metazoan-specific protein biogenesis at the ER. homeostasis. These findings suggest that covalent modification CELL BIOLOGY of RPL26 with UFM1 plays a key role in protein biogenesis in UFMylation | RPL26 | UFM1 | endoplasmic reticulum the early secretory pathway. Results biquitin fold modifier 1 (UFM1) is an 85-amino acid Uubiquitin-like protein modifier (UBL) that is ubiquitously Loss of UFMylation or De-UFMylation Impairs ER-Associated Protein expressed in metazoans but absent from fungi (1) and is essential Degradation. We identified the entire suite of genes encoding the for brain and hematopoietic development (2–4). Like ubiquitin and UFM1 system, including UFM1 itself and all known conjugation and other UBLs, UFM1 is covalently ligated to lysine residues on target deconjugation enzymes, in a genome-wide CRISPR/Cas9-mediated proteins by a three-step cascade catalyzed by E1, E2, and E3 en- gene knockout analysis of substrate-selective ER-associated protein degradation (ERAD) in human K562 erythroleukemia cells (26) zymes, encoded by UBA5, UFC1,andUFL1 genes, respectively (1, (Fig. 1B). ERAD is a quality control process by which folding- or 5) (Fig. 1A). UFM1 is cleaved from its targets by the UFM1- assembly- defective proteins are targeted for proteasome-mediated specific thiol protease, UFSP2 (5–7). Although the basic chemis- try of UFM1 conjugation is well understood, its physiological function is not. Homozygous disruption of the UFM1 pathway in Significance mice causes midgestation embryonic lethality associated with im- paired hematopoiesis (2, 8–10). In humans, hypomorphic loss-of- Ubiquitin fold modifier 1 (UFM1) is a ubiquitin-like post- function alleles of genes that code for UFM1, UBA5, or UFPS2 translational modifier that is essential for tissue development are linked to diverse pathologies, including leukodystrophy (3), in metazoans. Genetic ablation of UFM1 or the genes that epileptic encephalopathy (11), spinocerebellar ataxia (12), and encode the enzymes that conjugate UFM1 to protein targets skeletal abnormalities (13–15), revealing an essential role for causes endoplasmic reticulum (ER) stress, suggesting a role for UFMylation in multiple organ systems. Although several cellular this modification in ER protein homeostasis. Here, we show proteins have been reported to beUFMylationtargets(5,7,16– that RPL26, a largely uncharacterized ribosomal protein, is the 18), none of these have been demonstrated to form covalent ad- primary cellular target of UFMylation. Ribosomal RPL26 is ducts with UFM1 under physiological conditions and none have subject to a dynamic cycle of UFMylation and de-UFMylation, been plausibly linked to the cellular or organismal pathophysiol- catalyzed by enzymes that are tethered to the cytoplasmic ogies associated with loss-of-function UFMylation mutants in hu- surface of the ER. UFMylated RPL26 and the UFMylation ma- mans or experimental animals. chinery are in close proximity to the SEC61 translocon, sug- UFMylation is intimately linked to the secretory pathway. The gesting a role for UFMylation in protein biogenesis at the ER. UFM1-specific ligase, UFL1, is recruited to the cytosolic face of the endoplasmic reticulum (ER) membrane, likely through its sta- Author contributions: C.P.W., D.E.L., C.R., and R.R.K. designed research; C.P.W., D.E.L., ble association with the ER resident transmembrane protein, L.Z., C.R., R.Y.M., and P.A.D. performed research; C.P.W. and D.E.L. contributed new reagents/analytic tools; C.P.W., D.E.L., L.Z., C.R., P.A.D., N.T.I., J.E.E., and R.R.K. analyzed DDRGK1 (19). Moreover, UFMylation genes are transcriptional data; and C.P.W., D.E.L., and R.R.K. wrote the paper. targets of the unfolded protein response (UPR) and the UFM1 The authors declare no conflict of interest. promoter contains a putative binding site for the ER stress- responsive transcriptional activator XBP1 (20). Targeted disrup- This article is a PNAS Direct Submission. tion of genes encoding UFM1 or UFM1 conjugation machinery Published under the PNAS license. weakly activates an ER stress response in a subset of cell types in 1To whom correspondence should be addressed. Email: [email protected]. culture (21–23) and enhances ER stress-induced apoptosis in pan- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. creatic beta cells (24). Although the UFMylation pathway is not 1073/pnas.1816202116/-/DCSupplemental. essential for viability of cells in culture, the impaired hematopoeisis Published online January 9, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1816202116 PNAS | January 22, 2019 | vol. 116 | no. 4 | 1299–1308 Downloaded by guest on October 1, 2021 A B UFC1 Separate GFPhigh UBA5 Reporter +Cas9-BFP and GFPlow UBA5~ K562 cells +10 guide dox dox populations UBA5 ? CRKO library pulse washout by FACS UFM1 GlySerCys Gly UFC1~ target target low high reporter 1 high GFP GFP target UFL1 reporter 2 UFSP2 reporter 3 reporter 4 ODR4 CDK5RAP3 DDRGK1 Cytosol gene effect repeat with stabilizing hits C ... controls e A additional counts in GFP low reporters counts in GFP count sgRNAs gen genegene B gene D calculate gene effect by deep ER membrane compare gene effects from normalized sequencing log ratio of counts 8 C Reporter K562 A1ATNHK-GFP 6 WT UFM1KO GFP-RTAE177Q INSIG1-GFP emetine (h): 020.5 1 1.5 3020.5 1 1.5 3 u* 4 GFP CD147(CG) 37 37 GAPDH 2 -log(P-value) gene effect 1 0 3 100 WT 5 75 UFM1KO * −2 50 UFL1 UFC1 25 UBA5 UFM1 ODR4 AMFR remaining UFSP1 UFSP2 DERL2 RNF139 SYVN1 UBE2G2 0 DDRGK1 % CD147 (CG) 0123 HERPUD2 CDK5RAP3 emetine (h) UFMylation ERAD * U2OS 100 WT *** * D * * KO * UBA5 * WT UBA5KO UFSP2KO DDRGK1KO 75 UFSP2KO KO emetine (h):01 1 2 3 0 230 1230 123 50 DDRGK1 CD147(CG) 37 25 GAPDH 37 remaining % CD147 (CG) 0 0123 emetine (h) KO E KO UFSP2 + U2OS UFSP2 WT UFSP2 *** ** * WT C302S Y290H * DNA: empty vector UFSP2 UFSP2 UFSP2 100 empty vector * * C302S emetine (h):01 1 2 3 0 230 123 0 123 75 UFSP2 37 UFSP2Y290H CD147(CG) 50 GAPDH 37 remaining 25 % CD147 (CG) 0 0123 emetine (h) F HEK293 WT KO KO 100 n.s. WT UFM1 UFSP2 UFM1KO n.s. emetine (h):01 1 2 3 0 230 123 75 UFSP2KO CD147(CG) 37 50 GAPDH 37 remaining 25 % CD147 (CG) 0 0123 emetine (h) Fig. 1. Loss of UFMylation or de-UFMylation impairs ERAD. (A) The UFMylation system shown in relation to the ER membrane bilayer. Known UFM1 conjugation or deconjugation machinery is rendered in blue and orange, respectively. Interacting partners of unknown function are shown in white with interactions denoted by dashed lines. (B) Identification of the UFMylation pathway from functional genomic analysis of substrate-selective ERAD. (Top) Forward genetic screening pipeline. Four independent genome-wide screens were iteratively conducted with the indicated GFP-tagged reporters and the data combined into a “gene effect” metric. Complete details can be found in ref. 26. (Bottom) Bubble plot of gene effects for UFM1-related genes and a reference set of ERAD genes. Data are from table S1 in ref. 26. Each of the four ERAD reporters is represented by a different colored bubble; the diameter indicates the −log P value. SYVN1 encodes HRD1, AMFR encodes GP78, and RNF139 encodes TRC8. (C) UFM1 knockout impairs turnover of CD147(CG) in K562 cells. (Top) Cells were treated with the protein synthesis inhibitor, emetine, for the indicated times before lysis.
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