BLTP2 is enriched at ER contacts with the PM and PM-connected tubular internal membranes. (A) Domain diagram of human BLTP2 and of the internally tagged (Halo, EGFP, or 2xV5 epitopes) BLTP2. TM indicates the transmembrane region of BLTP2. (B) Schematic model of the arrangement of BLTP2 at contacts of the ER with other membranes. The arrow indicates the site where tags (Halo, EGFP, or 2xV5 epitopes) were inserted. Structures are predicted using AlphaFold3. (C and D) U2OS cells (C) and MDA-MB-231 cells (D) stably expressing BLTP2^Halo show enrichment of this protein at ER-PM contacts near the edge of the cell. An optical section close to the basal surface (see dashed red line in the cartoon) is shown. (E) HeLaM cells stably expressing BLTP2^Halo show enrichment of the protein at tubular structures. (F) Localization of endogenous BLTP2 in gene-edited HeLaM cells where the 2xV5 epitope was inserted in the coding sequence of BLTP2. Anti-V5 immunofluorescence reveals enrichment of BLTP2 on tubular structures. (G) Validation of the endogenous tagging of BLTP2 by western blotting. Anti-BLTP2 immunoprecipitation co-enriched a V5 immunoreactive band. (H) Endogenous BLTP2 (V5 immunoreactivity) co-localizes with exogenous BLTP2^Halo on the same tubular structures. (I) CLEM revealed that a BLTP2-positive tubular structure represents a tubular membrane surrounded by ER. Scale bar, 500 nm. (J) BLTP2^Halo localizes at the distal portion of tubular structures that are positive for the PM marker GFP-CAAX and are labeled by the membrane-impermeant PM dye CellBrite. (K) TMEM24-mCherry, another ER-PM contact protein, is also present on BLTP2-positive tubular structures but only partially co-localizes with BLTP2^Halo. (L) GFP-MAPPER, an artificial ER-PM tethering protein, is also present on BLTP2-positive tubular structures but does not overlap with BLTP2^Halo. (M) Schematic drawing of BLTP2 localization at contacts of the ER with both the outer PM and the distal portions of PM-connected tubular structures. Source data are available for this figure: SourceData F1.
Figure 1.

BLTP2 is enriched at ER contacts with the PM and PM-connected tubular internal membranes. (A) Domain diagram of human BLTP2 and of the internally tagged (Halo, EGFP, or 2xV5 epitopes) BLTP2. TM indicates the transmembrane region of BLTP2. (B) Schematic model of the arrangement of BLTP2 at contacts of the ER with other membranes. The arrow indicates the site where tags (Halo, EGFP, or 2xV5 epitopes) were inserted. Structures are predicted using AlphaFold3. (C and D) U2OS cells (C) and MDA-MB-231 cells (D) stably expressing BLTP2^Halo show enrichment of this protein at ER-PM contacts near the edge of the cell. An optical section close to the basal surface (see dashed red line in the cartoon) is shown. (E) HeLaM cells stably expressing BLTP2^Halo show enrichment of the protein at tubular structures. (F) Localization of endogenous BLTP2 in gene-edited HeLaM cells where the 2xV5 epitope was inserted in the coding sequence of BLTP2. Anti-V5 immunofluorescence reveals enrichment of BLTP2 on tubular structures. (G) Validation of the endogenous tagging of BLTP2 by western blotting. Anti-BLTP2 immunoprecipitation co-enriched a V5 immunoreactive band. (H) Endogenous BLTP2 (V5 immunoreactivity) co-localizes with exogenous BLTP2^Halo on the same tubular structures. (I) CLEM revealed that a BLTP2-positive tubular structure represents a tubular membrane surrounded by ER. Scale bar, 500 nm. (J) BLTP2^Halo localizes at the distal portion of tubular structures that are positive for the PM marker GFP-CAAX and are labeled by the membrane-impermeant PM dye CellBrite. (K) TMEM24-mCherry, another ER-PM contact protein, is also present on BLTP2-positive tubular structures but only partially co-localizes with BLTP2^Halo. (L) GFP-MAPPER, an artificial ER-PM tethering protein, is also present on BLTP2-positive tubular structures but does not overlap with BLTP2^Halo. (M) Schematic drawing of BLTP2 localization at contacts of the ER with both the outer PM and the distal portions of PM-connected tubular structures. Source data are available for this figure: SourceData F1.

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