Protease cleavage assay to assess the topology of shr3-35-NubA and gap1-2TM-Cub-GST. (A) Schematic diagram of the split ubiquitin shr3-35-NubA construct (pAR67) and possible topological orientations: I. proper topology, i.e., TM in native orientation with NubA exposed to the cytoplasm and Proteinase K (Prot K) sensitive; II. improper inverted topology, i.e., TM in non-native orientation with NubA oriented toward the lumen and Prot K insensitive in the absence of detergent (NP-40). The ER-membrane topology of inserted shr3-35-NubA was examined in intact microsomes isolated from strain FGY135 (gap1Δ shr3Δ) carrying pPL1257 (GAP1-Cub-GST) and pAR67 (shr3-35-NubA). Microsomes were incubated with Prot K for 2 h with or without the addition of 0.2% NP-40 as indicated. shr3-35 was detected with the use of α-Shr3 directed to a C-terminally located epitope. Kar2 served as a control for microsome integrity. (B) Schematic diagram of the split ubiquitin gap1-2TM-Cub-GST construct (pIM1) and possible topological orientations: I. proper topology, i.e., TM in native orientation with Cub-GST exposed to the cytoplasm and Proteinase K (Prot K) sensitive; II. improper inverted topology, i.e., TM in non-native orientation with Cub-GST oriented toward the lumen and Prot K insensitive in the absence of detergent (NP-40). The ER-membrane topology of inserted gap1-2TM-Cub-GST-6xHA was examined in intact microsomes isolated from strain HKY15 (ssy1Δ) carrying (gap1-2TM-Cub-GST) and incubated with Prot K for 2 h with or without the addition of 0.2% NP-40 as indicated. Kar2 served as a control for microsome integrity. Source data are available for this figure: SourceData FS5.