Fig. 3: Substrate C-tail samples EMC before translocation.
From: EMC rectifies the topology of multipass membrane proteins
a, The 23L-SQS reporter (top) consists of a short translocated N-tail, a first TMD made of 23 leucine residues, an ~100 amino acid cytosolic loop, a second TMD and flanking sequences from SQS and a short translocated C-tail. Both terminal tails have glycosylation sites to monitor translocation. 35S-methionine labeled 23L-SQS was translated in the presence of SPCs derived from ΔEMC cells or cells expressing variants of EMC3 (WT, Mcyt-1-S and R31A). Products with different glycosylation states are indicated. Quantification of three independent experiments (mean ± s.d.) is plotted. C-tail translocation is determined as the per cent of glycosylated products that contains two glycans. b, 35S-methionine-labeled 23L-SQS without or with a single cysteine within the C-tail, translated in the presence of SPCs derived from ΔEMC (ΔE) cells or cells expressing EMC3-216C (in which a single cysteine is introduced into cysteine-free EMC3 at residue 216 located in its cytosolic vestibule)6. One aliquot was analyzed directly (−BMH) and another was treated with BMH. Crosslinked samples were analyzed directly (+BMH) or after EMC3 denaturing immunoprecipitation via FLAG tag (+BMH EMC3 denat. IP). The positions of 23L-SQS with zero, one or two glycans, and crosslinks between 23L-SQS and EMC3 or lumenal proteins are indicated. Lanes containing EMC3 crosslinks were digested with PNGase F to confirm that the crosslinks contain either zero or one glycan and hence are not crosslinks to fully translocated double-glycosylated 23L-SQS. c, 35S-methionine-labeled 23L-SQS containing a single cysteine within the C-tail or the SQS TMD, translated in the presence of EMC3-216C SPCs and analyzed similarly to b. d, 35S-methionine-labeled 23L-SQS without or with a single Bpa incorporated within the C-tail (through an amber suppression system) was translated in the presence of SPCs derived from WT or ΔE cells. One aliquot was analyzed directly (−UV) and another was subjected to UV crosslinking. Crosslinked samples were analyzed after EMC3 denaturing immunoprecipitation via FLAG tag (+UV EMC3 denat. IP). Labeling is similar to b.
