Fig. 2.

Querying tail-anchored (TA) protein biogenesis at the endoplasmic reticulum (ER). (A) (Left) TA proteins can be inserted into the lipid bilayer by either the EMC or GET insertases. (Right) TAs containing a moderately hydrophobic transmembrane domain such as SEC61β can use either EMC or GET1/GET2 to insert, obscuring strong effects on insertion when obstructing only one of these partially redundant pathways. Therefore, use of an EMC2 fixed guide dual library should uncover defined epistatic relationships between factors in either the GET or EMC pathways. (B) Schematic of the split GFP reporter system used to assess insertion of SEC61β into the ER. K562 cells expressing CRISPRi machinery were engineered to constitutively express GFP1-10 in the ER lumen. The 11th β -strand of GFP is fused to the C-terminus of SEC61β, allowing for conjugation and fluorescence of the full GFP upon insertion into the ER membrane. RFP is expressed as a normalization marker, separated by a viral P2A sequence. (C) Depletion of EMC and GET pathway components in the SEC61β reporter cell line. The SEC61β cell line was separately transduced with dual guides targeting EMC2 alone, GET2 alone, EMC2 and GET2, or a non-targeting control. The GFP:RFP ratio, a measure of SEC61β insertion at the ER, is plotted for each dual guide