Interactions of BLTP2 with FAM102B and N-BAR domain proteins at PM-connected tubular endosomes in HeLaM cells. (A) Solo expression of FAM102A-GFP in HeLaM cells results in its diffuse localization, while solo expression of FAM102B-GFP results in its enriched localization on tubular structures similar to BLTP2 localization. (B and C) FAM102B-GFP co-localizes with either BLTP2^Halo (B) or endogenous BLTP2 (C) on tubular structures in HeLaM cells. (D–F) Co-expression of BLTP2^EGFP FL with either BLTP2^Halo FL or BLTP2^Halo-△FBM, respectively, in HeLaM cells, showing that deletion of the FBM motif decreases the enrichment of BLTP2 at the tubular structures (D). (E) shows the method used for the quantification. The quantification result is shown in F. Two-tailed t test. Mean ± SEM. (FL) n = 63 tubules from nine cells; (△FBM) n = 111 tubules from 15 cells. (G) GFP-FBP17 localizes on the same BLTP2^Halo-positive tubules but does not overlap with the BLTP2^Halo signal. (H and I) GFP-mAmph2 co-localizes with BLTP2^Halo on the tubular structures (H), while GFP-mAmph2-BAR* (I) localizes on the same tubules but does not overlap with BLTP2^Halo. (J) AlphaFold3 predicts the interaction of a loop of BLTP2 (loop1) with the SH3 domain of amphiphysin. This loop harbors a RAPHPP sequence (RxP motif), which fits an SH3 domain–binding consensus. (K and L) Co-expression of BLTP2^EGFP FL with a BLTP2^Halo construct in which each of these six aa of the SH3-binding consensus was mutated to alanine (BLTP2^HaloRP→AA), or BLTP2^HaloRP→AA with the additional deletion of the FBM (BLTP2^HaloRP→AA-△FBM), showing a synergistic effect of abolishing SH3 domain binding and FAM102 binding in reducing the targeting of BLTP2 to the tubules. The quantification result is shown in L. One-way ANOVA. Mean ± SEM. (FL) n = 63 tubules from nine cells; (RP→AA) n = 103 tubules from 13 cells; (RP→AA and △FBM) n = 97 tubules from 13 cells. (M) Schematic drawing depicting co-localization of BLTP2, FAM102B, and amphiphysin 2 on the PM-connected tubular endosomes, but segregation form FBP17.
Figure 6.

Interactions of BLTP2 with FAM102B and N-BAR domain proteins at PM-connected tubular endosomes in HeLaM cells. (A) Solo expression of FAM102A-GFP in HeLaM cells results in its diffuse localization, while solo expression of FAM102B-GFP results in its enriched localization on tubular structures similar to BLTP2 localization. (B and C) FAM102B-GFP co-localizes with either BLTP2^Halo (B) or endogenous BLTP2 (C) on tubular structures in HeLaM cells. (D–F) Co-expression of BLTP2^EGFP FL with either BLTP2^Halo FL or BLTP2^Halo-△FBM, respectively, in HeLaM cells, showing that deletion of the FBM motif decreases the enrichment of BLTP2 at the tubular structures (D). (E) shows the method used for the quantification. The quantification result is shown in F. Two-tailed t test. Mean ± SEM. (FL) n = 63 tubules from nine cells; (△FBM) n = 111 tubules from 15 cells. (G) GFP-FBP17 localizes on the same BLTP2^Halo-positive tubules but does not overlap with the BLTP2^Halo signal. (H and I) GFP-mAmph2 co-localizes with BLTP2^Halo on the tubular structures (H), while GFP-mAmph2-BAR* (I) localizes on the same tubules but does not overlap with BLTP2^Halo. (J) AlphaFold3 predicts the interaction of a loop of BLTP2 (loop1) with the SH3 domain of amphiphysin. This loop harbors a RAPHPP sequence (RxP motif), which fits an SH3 domain–binding consensus. (K and L) Co-expression of BLTP2^EGFP FL with a BLTP2^Halo construct in which each of these six aa of the SH3-binding consensus was mutated to alanine (BLTP2^HaloRP→AA), or BLTP2^HaloRP→AA with the additional deletion of the FBM (BLTP2^HaloRP→AA-△FBM), showing a synergistic effect of abolishing SH3 domain binding and FAM102 binding in reducing the targeting of BLTP2 to the tubules. The quantification result is shown in L. One-way ANOVA. Mean ± SEM. (FL) n = 63 tubules from nine cells; (RP→AA) n = 103 tubules from 13 cells; (RP→AA and △FBM) n = 97 tubules from 13 cells. (M) Schematic drawing depicting co-localization of BLTP2, FAM102B, and amphiphysin 2 on the PM-connected tubular endosomes, but segregation form FBP17.

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