Figure 5. USP13 regulates ubiquitination of Ubl4A A, Ubl4A but not Trc35 is ubiquitinated in cells.

(A) Ubl4A or Trc35 was immunoprecipitated from an extract of HEK293 cells under denaturing condition and blotted with the indicated antibodies. (B) Membrane-associated Ubl4A is preferentially ubiquitinated. HEK293 cells were fractionated into an ER-containing membrane and cytosol fractions. Ubl4A immunoprecipitated from these fractions was analyzed by immunoblotting. (C) Ubl4A is conjugated with Lys48-linked ubiquitin chains. Ubl4A immunoprecipitated from extracts of cells expressing the indicated ubiquitin variants was analyzed by immunoblotting. (D and E) USP13 depletion causes accumulation of ubiquitinated Ubl4A in cells. (D) Ubiquitinated Ubl4A was analyzed in cells expressing the indicated shRNAs and HA-ubiquitin. (E) Ubiquitinated Ubl4A was analyzed in control wild-type cells and USP13 knockout (−/−) cells expressing HA-ubiquitin. (F) USP19 depletion does not affect Ubl4A ubiquitination. Asterisk indicates a non-specific band. (G) In vitro deubiquitination of Ubl4A by USP13. Ubl4A purified from USP13 knockout cells was treated with either buffer or increased concentration of FLAG-USP13 (0.15 µM–0.6 µM) at 37°C for 2 hr. Where indicated, Ub-aldehyde (Ub-Al, 1 µM) was included. (H) TCRα purified from USP13 knockout cells was incubated with either buffer or FLAG-USP13 (0.15 µM–0.6 µM) at 37°C for 2 hr.

DOI: http://dx.doi.org/10.7554/eLife.01369.010

Figure 5—figure supplement 1. Regulation of Ubl4A by ubiquitination.

(A) USP13 does not regulate ubiquitination of Bag6. HEK293 cells were transfected with the indicated shRNA constructs. Endogenous Bag6 was immunoprecipitated under denaturing condition and was analyzed by immunoblotting. Where indicated, a fraction of the lysate (input) was analyzed directly by immunoblotting. (B) The proteasome inhibitor MG132 does not increase the steady state level of Ubl4A. Immunoblotting analysis of whole cell extracts from HEK293 cells either untreated or treated with MG132 (20 µM, 6 hr). The numbers show the quantification of Ubl4A levels. Note that MG132 stabilizes the transcription factor ATF4, but does not affect Ubl4A protein level. (C) Ubiquitinated TCRα accumulates in USP13 knockdown cells. (D) The USP13 activity is increased at pH 8.0. Purified FLAG-USP13 (120 nM) was incubated with Ub-AFC (2.5 µM) under the indicated pH condition. (E) Ubl4A purified from USP13 knockout cells was incubated with increased amount of GST-USP13 at 37°C for 2 hr.

DOI: http://dx.doi.org/10.7554/eLife.01369.011