Figure 4.
An interaction of BLTP2 with FAM102A and FAM102B enriches BLTP2 at ER-PM contacts. (A and B) Interaction diagram exported from the Yeast Interactome Website (https://yeast-interactome.biochem.mpg.de:3838/interactome/) revealing protein interactions of the BLTP2 yeast orthologs Fmp27 (A, score = 10) and Ypr117w (B, score = 3) with Ybl086c, the ortholog of mammalian FAM102A/B. (C) Domain organization of Ybl086c with FAM102A/B comprising an N-terminal C2 domain and a C-terminal disordered region. The AlphaFold3-predicted BLTP-binding site in FAM102A and B is shown in J (see also Fig. S3 A). (D) Solo expression of FAM102A-GFP shows localization at the PM in U2OS cells, with no focal accumulations as expected for ER-PM contact sites. (E) In U2OS cells co-expressing FAM102A-GFP and BLTP2^Halo, the two proteins co-localize at ER-PM contacts. (F) In COS-7 cells, where BLTP2 does not accumulate at ER-PM contacts when expressed alone, co-expression of FAM102A-GFP recruits BLTP2^Halo to ER-PM contacts. (G) Solo expression of FAM102B-GFP in U2OS cells results in its diffuse localization. (H) In U2OS cells co-expressing FAM102B-GFP and BLTP2^Halo, the two proteins co-localize at ER-PM contacts. (I) In COS-7 cells, where BLTP2 does not accumulate at ER-PM contacts when expressed alone, co-expression of FAM102B-GFP recruits BLTP2^Halo to ER-PM contacts. (J) AlphaFold3 predicts an interaction between the C-terminal region of FAM102A (magenta) and a two-helix hairpin (green) (Fam102-binding motif, FBM) projecting out of the BLTP2 rod-like core (blue). (K) Left: COS-7 cells co-expressing BLTP2^EGFP and BLTP2^Halo-△FBM with FAM102A-iRFP showing that BLTP2^Halo-△FBM is not co-enriched with WT BLTP2^EGFP at ER-PM contact sites but remains diffuse throughout the ER. Right: Intensity profile of the red dashed line in the EGFP and Halo channels of the images at left shows the enrichment of BLTP2^EGFP (green) over BLTP2^Halo-△FBM (red) at PM patches. (L) The enrichment ratio of BLTP2^EGFP and BLTP2^Halo-△FBM at the basal surface when co-expressed with FAM102A-iRFP or FAM102B-iRFP are quantified. n = 15 cells for co-expressing FAM102A-iRFP and n = 9 cells for co-expressing FAM102B-iRFP. Mean ± SEM. (M) Top: Diagram of the FAM102B-EGFP and the SSPB-FAM102B-IDR-EGFP constructs. Bottom: Schematic drawing of the improved light-induced dimerization system to recruit FAM102B-IDR to the lysosomal membrane. (N) COS-7 cells expressing LAMP1-mCh-iLID show recruitment of SSPB-FAM102B-IDR-EGFP to LAMP1-positive organelles upon blue light activation, with only BLTP2-FL being co-recruited, but not BLTP2-ΔFBM.

An interaction of BLTP2 with FAM102A and FAM102B enriches BLTP2 at ER-PM contacts. (A and B) Interaction diagram exported from the Yeast Interactome Website (https://yeast-interactome.biochem.mpg.de:3838/interactome/) revealing protein interactions of the BLTP2 yeast orthologs Fmp27 (A, score = 10) and Ypr117w (B, score = 3) with Ybl086c, the ortholog of mammalian FAM102A/B. (C) Domain organization of Ybl086c with FAM102A/B comprising an N-terminal C2 domain and a C-terminal disordered region. The AlphaFold3-predicted BLTP-binding site in FAM102A and B is shown in J (see also Fig. S3 A). (D) Solo expression of FAM102A-GFP shows localization at the PM in U2OS cells, with no focal accumulations as expected for ER-PM contact sites. (E) In U2OS cells co-expressing FAM102A-GFP and BLTP2^Halo, the two proteins co-localize at ER-PM contacts. (F) In COS-7 cells, where BLTP2 does not accumulate at ER-PM contacts when expressed alone, co-expression of FAM102A-GFP recruits BLTP2^Halo to ER-PM contacts. (G) Solo expression of FAM102B-GFP in U2OS cells results in its diffuse localization. (H) In U2OS cells co-expressing FAM102B-GFP and BLTP2^Halo, the two proteins co-localize at ER-PM contacts. (I) In COS-7 cells, where BLTP2 does not accumulate at ER-PM contacts when expressed alone, co-expression of FAM102B-GFP recruits BLTP2^Halo to ER-PM contacts. (J) AlphaFold3 predicts an interaction between the C-terminal region of FAM102A (magenta) and a two-helix hairpin (green) (Fam102-binding motif, FBM) projecting out of the BLTP2 rod-like core (blue). (K) Left: COS-7 cells co-expressing BLTP2^EGFP and BLTP2^Halo-△FBM with FAM102A-iRFP showing that BLTP2^Halo-△FBM is not co-enriched with WT BLTP2^EGFP at ER-PM contact sites but remains diffuse throughout the ER. Right: Intensity profile of the red dashed line in the EGFP and Halo channels of the images at left shows the enrichment of BLTP2^EGFP (green) over BLTP2^Halo-△FBM (red) at PM patches. (L) The enrichment ratio of BLTP2^EGFP and BLTP2^Halo-△FBM at the basal surface when co-expressed with FAM102A-iRFP or FAM102B-iRFP are quantified. n = 15 cells for co-expressing FAM102A-iRFP and n = 9 cells for co-expressing FAM102B-iRFP. Mean ± SEM. (M) Top: Diagram of the FAM102B-EGFP and the SSPB-FAM102B-IDR-EGFP constructs. Bottom: Schematic drawing of the improved light-induced dimerization system to recruit FAM102B-IDR to the lysosomal membrane. (N) COS-7 cells expressing LAMP1-mCh-iLID show recruitment of SSPB-FAM102B-IDR-EGFP to LAMP1-positive organelles upon blue light activation, with only BLTP2-FL being co-recruited, but not BLTP2-ΔFBM.

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