Fig. 4: TPSN assembly and degradation depends on evolutionary conserved charge in its transmembrane domain.

a Reduced TPSN steady-state levels in TAP1 depleted HEK293 cells depend on the RNF185/MBRL complex. RNF5, RNF185 and MBRL were deleted in TAP1 knockout cells. Cells were lysed, and protein extracts were subjected to SDS–PAGE followed by immunoblotting with the indicated antibodies. Parental cells were used as control. b TPSN steady-state levels are increased by overexpression of its binding partner TAP1. Parental and MBRL KO cells expressing TPSN-sfGFP-3xHA were transduced with a TAP1 overexpressing vector or an empty vector. TPSN-sfGFP-3xHA levels were assessed by flow cytometry (based on GFP fluorescence). c TPSN stability is increased by overexpression of its binding partner TAP1. Degradation of TPSN was analysed after inhibition of protein synthesis by cycloheximide (CHX) in Parental and MBRL KO cells expressing TPSN-sfGFP-3xHA in the presence and absence of a TAP1 overexpressing vector. Cell extracts were resolved by SDS–PAGE and subjected to immunoblotting for the proteins indicated. Quantifications for TPSN-sfGFP-3xHA and overexpressed TAP1 protein levels of 3 independent experiments are shown in the graph below as mean; error bars represent the standard deviation. d Schematic representation of the interaction between TAP1/2 and TPSN mediated through an intramembrane salt bridge as described by Blees et al. (Reference #16). The lysine at position 428 of TPSN forms a salt bridge with an aspartic acid of TAP1 and TAP2, at position 32 and 16, respectively. e Degradation of TPSN and TPSN K428A was examined after inhibition of protein synthesis by cycloheximide (CHX). Cell extracts were analysed by SDS–PAGE and immunoblotting. TPSN and TPSN K428A were detected with anti-HA antibodies. GAPDH was used as a loading control and detected with an anti-GAPDH antibody. The graph shows the average of 3 experiments as the mean; error bars represent the standard deviation. f Immunoprecipitation of TPSN-sfGFP-3xHA and the indicated mutants. Cells expressing the indicated HA-tagged constructs were lysed in a buffer containing 1% DMNG, subjected to immunoprecipitation using anti-HA beads. Proteins were eluted and subjected to SDS–PAGE, followed by immunoblotting with the indicated antibodies.