Fig. 6: TM propensity of the Ybr196c-a protein.

a Ybr196c-a colocalizes at the ER with Scs2-TM. Chromosomally integrated mCherry-Scs2-TM and plasmid-borne Ybr196c-a-EGFP localization was assessed using confocal microscopy. White line is scale bar = 2μ. b Ybr196c-a colocalizes at the ER with Sec13. Chromosomally integrated Sec13-RFP and plasmid-borne Ybr196c-a-EGFP localization was assessed using confocal microscopy. White line is scale bar = 2μ. c Membrane association assay. Cell lysates were fractionated by centrifugation into a cytosolic fraction in the supernatant (S1) or a pelleted membrane fraction (P1). The pellet was suspended in either lysis buffer (LB) and centrifuged to generate S2 and P2 fractions, or buffer containing 6M urea, which solubilizes peripheral membrane proteins, to generate S3 and P3 fractions. Fractions were then subjected to SDS-PAGE and immunoblotted with anti-GFP (to detect Ybr196c-a), anti-Sec61 (an integral ER-membrane protein), and anti-Pdi1 (an ER luminal protein) antibodies. A dot indicates the full-length Ybr196c-a-EGFP fusion protein and the bands of lower MW are degradation products of this fusion. A star indicates a spurious soluble protein of higher MW than Sec61 that is recognized non-specifically by the anti-Sec61 antibody. Primary uncropped blots are provided in the Source Data file. d Carbonate extraction assay. Cellular membranes were treated with a buffer control (S1/P1), Na₂CO₃ (S2/P2) to extract peripherally associated membrane proteins or luminal proteins, such as the ER luminal protein Pdi1, or 1% SDS (S3/P3) to at least partially solubilize integral membrane proteins, such as the Sec61 integral membrane protein control. Integral membrane proteins such as Sec61 and Ybr196c-a remain in the pellet fraction post carbonate treatment (P2), unlike soluble proteins like Pdi1 which shift to the solubilized supernatant (S2). Fractions were assessed by immunoblotting as in Fig. 6c. Primary uncropped blots are provided in the Source Data file.