Fig. 3: TPSN is a substrate of the RNF185/MBRL complex.

a TPSN levels are increased in RNF185 and MBRL knockout cells. Clonal RNF185, MBRL, RNF5, and HRD1 knockout lines were established from Flp-In T-Rex HEK293 cells expressing TPSN-sfGFP-3xHA. TPSN-sfGFP-3xHA levels were assessed by flow cytometry (based on GFP fluorescence). b RNF185/MBRL complex is essential for TPSN turnover. Degradation of TPSN was analysed in parental, RNF5, RNF185, and MBRL KO Flp-In T-Rex HEK293 cells expressing TPSN-sfGFP-3xHA after inhibition of protein synthesis by cycloheximide (CHX). Cell extracts were analysed by SDS–PAGE and immunoblotting. TPSN was detected with an anti-HA antibody. GAPDH was used as a loading control and detected with an anti-GAPDH antibody. Quantifications of 3 independent experiments are shown in the graph below as mean; error bars represent the standard deviation. c RNF185/MBRL complex promotes TPSN ubiquitination. TPSN-sfGFP-3xHA was immunoprecipitated from parental cells or cells lacking the indicated proteins and analysed by SDS–PAGE followed by immunoblotting with anti-HA and anti-ubiquitin antibodies. Parental cells were used as a negative control. d The steady-state levels of TPSN 4K-to-A tail mutant are high and largely unresponsive to ERAD inhibition. Flp-In T-Rex HEK293 cells expressing GFP-HA-tagged WT or 4K-to-A tail mutant TPSN were analysed by flow cytometry (based on GFP fluorescence) in the absence or presence of the p97 inhibitor CB-5083 (2.5 µM). e RNF185/MBRL complex promotes the ubiquitination of lysines in the cytosolic tail of TPSN. WT or 4K-to-A tail mutant TPSN expressed as GFP-HA fusion proteins were immunoprecipitated from the indicated cell lines and analysed by SDS–PAGE followed by immunoblotting with anti-HA and anti-ubiquitin antibodies. Parental cells were used as a negative control. f MBRL binds TPSN independently of its partners RNF185, TMUB1/2. Endogenous MBRL or HRD1 were immunoprecipitated from the indicated cells and precipitated proteins were analysed by immunoblotting with anti-HA (for TPSN-sfGFP-3xHA), anti-HRD1 and anti-SEL1L antibodies.