Figure 2.
Identification of TMEM55B as a Tex2 adaptor on LE/Lys. (A) Volcano plot of protein candidates coIPed with Tex2 in HEK293 cells compared with protein candidates coIPed with GFP tag only. Candidates that were considered significant (−log [P value] > 1.3; P < 0.05) were labeled in red (Log2 [fold change] > 0; increased in abundance) or blue (Log2 [fold change] < 0; decreased in abundance). (B) GFP-Trap assays demonstrate an interaction between GFP-Tex2 and Halo-TMEM55B in COS7 cells. (C and D) Representative wide-field images of live COS7 cells expressing either GFP-Tex2 (green; C) or GFP-E-syt1 (green; D) and Halo-TMEM55B with two boxed regions showing at the bottom. (E and F) Representative images of a live COS7 cell expressing GFP-Tex2 (green), Halo-TMEM55B (magenta; E) or Halo-TMEM55A (magenta; F), ER-tagRFP (red), and Lamp1-mCh (blue; a LE/lys marker) with two boxed regions showing at the bottom. (G) Pearson’s correlation coefficient of Tex2 vs. Lamp1 in absence of TMEM55 (11 cells) or upon TMEM55A (10 cells) or TMEM55B (13 cells) overexpression (>3 independent experiments). Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (H and I) Representative live-cell images of a GFP-Tex2-KI (green) cell without (H) or with expressing Halo-TMEM55B (I) and Lamp1-mCh (blue) with two boxed regions at the bottom. Yellow arrows denote the specific enrichment of GFP-Tex2-KI at LE/lys extensively contacting the ER. (J) Representative 3D rendering of a COS7 cell expressing GFP-Tex2 (green), Halo-TMEM55B (magenta), and Lamp1-mCh (blue) with y-z projection to the left and x-z projection to the right. (K) COS7 cells expressing GFP-Tex2, Halo-TMEM55B, and stained by DAPI, were fixed and imaged by 3D microscopy (left panel) and processed for TEM (middle panel). Fluorescence and TEM images were correlated (right panel) with two insets showing tight associations between the ER (green) and LE/lys (magenta). Scale bar, 10 μm in the whole cell images and 2 μm in the insets in C, D, E, F, and H–J; 5 μm in the whole cell image, and 1 μm in the insets in K. Source data are available for this figure: SourceData F2.

Identification of TMEM55B as a Tex2 adaptor on LE/Lys. (A) Volcano plot of protein candidates coIPed with Tex2 in HEK293 cells compared with protein candidates coIPed with GFP tag only. Candidates that were considered significant (−log [P value] > 1.3; P < 0.05) were labeled in red (Log2 [fold change] > 0; increased in abundance) or blue (Log2 [fold change] < 0; decreased in abundance). (B) GFP-Trap assays demonstrate an interaction between GFP-Tex2 and Halo-TMEM55B in COS7 cells. (C and D) Representative wide-field images of live COS7 cells expressing either GFP-Tex2 (green; C) or GFP-E-syt1 (green; D) and Halo-TMEM55B with two boxed regions showing at the bottom. (E and F) Representative images of a live COS7 cell expressing GFP-Tex2 (green), Halo-TMEM55B (magenta; E) or Halo-TMEM55A (magenta; F), ER-tagRFP (red), and Lamp1-mCh (blue; a LE/lys marker) with two boxed regions showing at the bottom. (G) Pearson’s correlation coefficient of Tex2 vs. Lamp1 in absence of TMEM55 (11 cells) or upon TMEM55A (10 cells) or TMEM55B (13 cells) overexpression (>3 independent experiments). Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (H and I) Representative live-cell images of a GFP-Tex2-KI (green) cell without (H) or with expressing Halo-TMEM55B (I) and Lamp1-mCh (blue) with two boxed regions at the bottom. Yellow arrows denote the specific enrichment of GFP-Tex2-KI at LE/lys extensively contacting the ER. (J) Representative 3D rendering of a COS7 cell expressing GFP-Tex2 (green), Halo-TMEM55B (magenta), and Lamp1-mCh (blue) with y-z projection to the left and x-z projection to the right. (K) COS7 cells expressing GFP-Tex2, Halo-TMEM55B, and stained by DAPI, were fixed and imaged by 3D microscopy (left panel) and processed for TEM (middle panel). Fluorescence and TEM images were correlated (right panel) with two insets showing tight associations between the ER (green) and LE/lys (magenta). Scale bar, 10 μm in the whole cell images and 2 μm in the insets in C, D, E, F, and H–J; 5 μm in the whole cell image, and 1 μm in the insets in K. Source data are available for this figure: SourceData F2.

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