Fig 2: The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus.(A) The catalytic activity of UCPWT (0.2 µg) and truncated UCP mutants (UCPN-term, UCPCore, UCPC-term, UCP?N and UCP?C) (0.2 µg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 µg) and HA-Ubiquitin (2 µg), treated with 10µM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCPWT (0.2 µg) and truncated UCP mutants (UCPC-term and UCP?C, 0.2 µg) were mixed with His-VHL (2 µg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 µg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 µg) were co-transfected into HEK-293T cells and treated with/without 10 µM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.

Fig 3: UBA52 is required for embryonic development.(A) Diagram of human ubiquitin genes and their chromosomal locations. (B) Schematic representation of the gene-targeting construct and screening strategy. Southern blots of EcoRI- and PstI-digested genomic DNA from embryonic stem cells showing the targeted allele. The probe was specific for exon 1. (C) Polymerase chain reaction (PCR) analysis of genomic DNA obtained from the tails of wild-type (Uba52+/+) and mutant (Uba52tm1a/+) mice using the PCR primers indicated. (D) Quantitative real-time reverse-transcription PCR of Uba52 mRNA expression in tissues (kidney, spleen, small intestine, and colon) of 3-week-old mice. mRNA levels were normalized to Actb levels. Uba52+/+ (n = 8), Uba52tm1a/+ (n = 8). Error bars indicate standard deviations. N.S. indicates not significant by two-tailed Student’s t-test. (E) Numbers of surviving offspring from Uba52tm1a/+ parents. Pictures show embryonic day (E) 10.5 and E13.5 littermates. Parentheses: expected numbers based on a Mendelian ratio.

Fig 4: RPS27a ubiquitination is altered by interference with mRNA translation.(A) Extracts from HEK293 WT and USP16 KO cells were separated on a linear 10–45% sucrose gradient by centrifugation. Input and gradient fractions were analyzed by immunoblotting using the indicated antibodies. (B) Extracts from HeLa WT (black line) and USP16 KO (red line) cells were separated on a linear 10–45% sucrose gradient by centrifugation and analyzed by polysome profiling. (C) HeLa WT and USP16 KO cells were treated with cycloheximide (CHX; 100 µg/ml), silvestrol (1 µM) or DTT (5 mM) for the specified times and cell extracts were analyzed by immunoblotting using the indicated antibodies. Efficiency of the treatments was assessed by immunoblotting showing rapid loss of short-lived cMYC after CHX and silvestrol treatments, and accumulation of the UPR-induced transcription factor ATF4 after DTT treatment. (D) HeLa WT and USP16 KO cells were treated with G418 (400 µg/ml) for 24 hr and cell extracts were analyzed by immunoblotting using the indicated antibodies (left panel). The signal of three independent experiments was quantified and the percentage of ubiquitinated RPS27a (% UbRPS27a) of total RPS27a (modified plus unmodified RPS27a) was determined using an Odyssey (LI-COR) imaging system (mean ± SD; right panel). Values below the left panel indicate the mean of three independent experiments. **p=0.01 (unpaired t-test).

Fig 5: PEX4 builds K48-linked ubiquitin chains and pex4-1 (pex4P123L) displays altered ubiquitination activity in vitro. (A–C). Ubiquitination assays containing recombinant human ubiquitin-activating enzyme (E1), A. thaliana UBC (either PEX4-PEX22111-283 or pex4P123L-PEX22111-283), and A. thaliana ubiquitin (Ub) were initiated by ATP addition and stopped at the indicated timepoints. The resulting samples were analyzed by SDS-PAGE followed by Coomassie staining (A) or immunoblotting with a-Ub (B) or a-PEX4 (C) antibodies. This experiment was conducted three times and representative results are shown. Ub, ubiquitin; Ub2, diubiquitin. (D–E). Accumulation of various ubiquitinated proteins. Graphs display quantification of signals detected by the a-Ub (D) or a-PEX4 (E) antibodies in the indicated regions of the immunoblots (B,C). Each point represents the quantification from a technical replicate at the indicated timepoint. The accumulation rate was calculated for each replicate, and the average rate is plotted. Significance was determined by two-tailed t-tests comparing the accumulation rates from pex4P123L-PEX22111-283 to PEX4-PEX22111-283. Brackets indicate significant differences between PEX4-PEX22111-283 and pex4P123L-PEX22111-283 rates (*p < 0.05; **p < 0.005). (F–H). Ubiquitination assays as in panels A–C except that various ubiquitin variants were used: A. thaliana Ub (wt), human Ub with all lysine residues substituted with arginine except K48 (K48 only), or human Ub with K48 substituted with arginine (K48R). Samples were analyzed by SDS-PAGE followed by Coomassie staining (F) or immunoblotting with a-Ub (G) or a-PEX4 (H) antibodies.