Fig 1: RHBDL4 interacts with ubiquitinated TMED7 and cleavage is specified by the TMED7 luminal domain.a HEK293T cells transiently transfected with FLAG-TMED7 and either empty vector (-), GFP-tagged RHBDL4 (R4-GFP) wt, the catalytically inactive SA mutant or the SA-UIM double mutant (SAUM) were lysed with Triton X-100 and subjected to GFP-specific immunoprecipitation (IP). Western blot (WB) analysis reveals increased co-purification of glycosylated (black arrow) and unglycosylated (circle) FLAG-TMED7, including several ubiquitinated forms (gray arrow) but not the 22-kDa cleavage fragment (open arrow) (n = 3). b HEK293T cells transfected with siRNA targeting either gp78 (sigp78) or with control siRNA (-) and either transiently transfected with FLAG-TMED7 and either empty vector (-) or the GFP-tagged RHBDL4 catalytically inactive SA mutant were lysed with Triton X-100 and subjected to GFP-specific IP. WB analysis reveals decreased co-purification of FLAG-TMED7. Quantification of ubiquitinated TMED7 in sigp78-treated cells relative to control is shown (means ± SEM, n = 3; **p < 0.01, two-sided Student’s t test, p = 0.0043). c Outline of FLAG-TMED7 highlighting the 20 amino acid-cleavage site-region. Below an outline of FLAG-TMED9, including the detailed sequence homologous to the displayed TMED7 sequence. Hexagon, position of N-linked glycan. SPase, site of predicted signal peptide cleavage; TMD, transmembrane domain. d HEK293T cells were transfected with either FLAG-TMED7, FLAG-TMED9 or chimera of the two. Chimera are labeled according to the following scheme, indicating the source protein for the respective part: luminal_TMD_cytoplasmic. In addition, for each substrate construct either empty vector (-) or HA-R4 was transfected. Cells were treated with MG132 (2 µM). Only for constructs with a TMED7 luminal domain a cleavage fragment (open arrow) becomes visible (n = 3). e Same experiment as in (d) with chimera constructs as indicated. Only for constructs containing the TMED7 amino acid sequence 135-154 a cleavage fragment (open arrow) becomes visible (n = 3). f HEK293T cells were transfected with either FLAG-TMED7 wt or a SA149PP mutant of TMED7 together with HA-R4 as indicated. Cleavage of FLAG-TMED7-SA149PP is reduced compared cleavage of wt FLAG-TMED7. Cells were treated with MG132 (2 µM), (means ± SEM, n = 4, **p < 0.01, two-sided Student’s t test, p = 0.0019). Source data are provided as a Source Data file.