FIGURE 1.

Mannose trimming and EDEM1 are required for ERAD of H2a. A, HEK 293 cells cotransfected with an H2a cDNA encoding vector were pulse-labeled for 20 min with [³⁵S]cysteine and chased for 0 or 5 h in complete medium in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4) of Kif (100 μm). After the pulse (0 h chase) or chase periods, the cells were lysed, H2a was immunoprecipitated, and the immunoprecipitates were separated in 12% SDS-PAGE followed by phosphorimaging. Bands corresponding to the H2a precursor and the naturally occurring cleaved fragment are indicated on the left. For the pulse samples, two bands can be seen for the H2a precursor and also for the fragment, with the lower ones corresponding to underglycosylated species (one of the three glycosylation sites unoccupied). B and C, similar to the pulse-chase experiment in A but performed with untreated cells transiently cotransfected with an H2a cDNA encoding vector together with either a GFP-containing pSUPER-retro vector (B and C, lanes 1 and 2) or with pSUPER encoding anti-EDEM1 shRNA (B, lanes 3 and 4) or with an EDEM1-HA containing pCMVsport2 plasmid (C, lanes 3 and 4). D, the bar graph shows the average percent H2a remaining after chase relative to the corresponding pulse, calculated from phosphorimaging quantitations of all H2a species (precursor and fragment) from three independent experiments similar to each of the ones shown in A–C. Error bars indicate S.D. between experiments. Student's t test renders p < 0.02 for values in all chase samples compared with control chase. E, in parallel with B, HEK 293 cells were transfected with either pSUPER (control, lane 1) or the same plasmid encoding anti-EDEM1 shRNA (lane 2). RNA was extracted 48 h post-transfection and used for RT-PCR with primers for EDEM1 mRNA (upper panel) compared with GAPDH (lower panel). Lane 3 shows a sample with no RNA template.