Article Title: Regulated assembly of the ER membrane protein complex
Figure Lengend Snippet: ( A ) Topology of the EMC subunits based on the structure of the human EMC . Note that EMC8 and 9 are paralogs and their binding to EMC2, and thus incorporation into the EMC, is mutually exclusive. ( B ) The stability of soluble and membrane-embedded EMC subunits is interdependent in cells. HeLa cells were treated with scrambled, EMC2, or EMC5 siRNA and harvested for Western blotting with the indicated antibodies. Similar results were observed in HEK293T, U2OS, and RPE1 cell lines and has been reported in previous studies ( ; ). ( C ) Soluble EMC2 stably recruits an E3 ubiquitin ligase. An in vitro translation reaction of 35 S-methionine-labeled 3xFLAG-EMC2 was fractionated on a 10-50% (w/v) sucrose gradient. Unfractionated translation (Total) and all fractions were subjected to FLAG-IP and subsequently incubated with ATP, His-tagged ubiquitin (His-Ub), as well as E1 and E2 enzymes to detect the presence of co-purifying E3 ligase activity. Ubiquitinated species were enriched after elution from the FLAG resin by Ni 2+ -chelate affinity chromatography. ( D ) E. coli -purified EMC2 is a monodisperse, soluble protein. His 14 - bd NEDD8-tagged EMC2 was purified on a Superdex 200 Increase 10/300 size-exclusion chromatography column. UV absorbance at 280 nm was monitored throughout the purification. ( E ) WNK1 co-purifies with EMC2 from human cells. HEK293T cell lines stably expressing GFP-EMC2 were used for purification of GFP-tagged EMC2 and its interacting proteins under native conditions using a biotinylated Avi-SUMO Eu1 -tagged anti-GFP nanobody (see methods). Samples of total lysate and eluate were analyzed by Western blot with anti-GFP and anti-WNK1 antibodies. ( F ) Endogenous NKCC1 expression relies on a functional EMC. HeLa cells were treated with either scrambled, EMC2, or EMC5 siRNA and harvested for Western blotting with the indicated antibodies. Expression of endogenous AE2, a previously identified EMC substrate, displays a similar dependence on the EMC for expression. ( G ) NKCC1 is post-translationally destabilized in the absence of the EMC. HEK293T cells stably expressing GFP-NKCC1-2A-RFP were treated with either scrambled, EMC2, or EMC5 siRNA and the GFP:RFP ratio was analyzed by flow cytometry. The observed post-translational destabilization of NKCC1 upon EMC depletion is consistent with a requirement for EMC in NKCC1 biogenesis.
Article Snippet: The human NKCC1 coding sequence was derived from Addgene plasmid # 49077, which was a gift from Biff Forbush.
Techniques: Binding Assay, Western Blot, Stable Transfection, In Vitro, Labeling, Incubation, Activity Assay, Affinity Chromatography, Purification, Size-exclusion Chromatography, Expressing, Functional Assay, Flow Cytometry