Fig. 4: Determinants of substrate C-tail translocation.
From: EMC rectifies the topology of multipass membrane proteins
a, 35S-methionine-labeled 23L-SQS variants with the different C-tail lengths indicated (top) translated in the presence of SPCs derived from WT or ΔE cells. Where indicated, 2 µM the Sec61 inhibitor ApraA was included in the translation reaction. Translation reactions were analyzed directly, and substrates with zero, one or two glycans attached are indicated. The two-glycan product is indicative of successful C-tail translocation. C-tail translocation was quantified by calculating the percentage of all glycosylated products that contain two glycans. b, 35S-methionine-labeled 23L-SQS variants with charged residue mutations in the C-tail flanking the TMD were analyzed as in a. The sequences of the variants are shown with the net C-tail flanking charge indicated. c, 35S-methionine-labeled 23L-SQS variants with leucine mutations in the TMD were analyzed as in a. The sequences of the variants are shown along with the hydrophobicity of each TMD as a ΔGapp score65, in which negative values indicate favored membrane insertion.
