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. 2019 Nov 5;116(45):22619-22623.
doi: 10.1073/pnas.1913509116. Epub 2019 Oct 21.

PDZD8 mediates a Rab7-dependent interaction of the ER with late endosomes and lysosomes

Andrés Guillén-Samander  1   2   3   4 , Xin Bian  1   2   3   4 , Pietro De Camilli  5   2   3   4   6
Affiliations

PDZD8 mediates a Rab7-dependent interaction of the ER with late endosomes and lysosomes

Andrés Guillén-Samander et al. Proc Natl Acad Sci U S A. .

Abstract

Contacts between the endoplasmic reticulum (ER) and other membranes are hot spots for protein-mediated lipid transport between the 2 adjacent bilayers. Compiling a molecular inventory of lipid transport proteins present at these sites is a premise to the elucidation of their function. Here we show that PDZD8, an intrinsic membrane protein of the ER with a lipid transport module of the SMP domain family, concentrates at contacts between the ER and late endosomes/lysosomes, where it interacts with GTP-Rab7. These findings suggest that PDZD8 may cooperate with other proteins that function at the ER-endo/lysosome interface in coordinating endocytic flow with lipid transport between endocytic membranes and the ER.

Keywords: SMP domain; lipid-transfer protein; membrane contact sites.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Localization of PDZD8 at MCS between the ER and late endosomes/lysosomes. (A) Domain organization of human PDZD8. (B) Confocal images of Cos7 cells showing that PDZD8-EGFP is localized throughout the ER but enriched at the ER surrounding a small subset of Lamp1-RFP–positive vesicles (arrows). (C) The expression of mCherry-Rab7a sequesters the bulk of PDZD8 on all mCherry-Rab7a–positive vesicles, producing a striking colocalization. (D) Percentage of cells with PDZD8-EGFP signal surrounding vesicles with and without mCherry-Rab7a coexpression. (E) Number of vesicles surrounded by PDZD8-EGFP per cell. Cells not expressing mCherry-Rab7a and without such vesicles were excluded from the count. Data for D and E represent mean ± SD, n = 3 experiments. **P = 0.004; ****P < 0.0001 (2-tailed, unpaired t test). (F) Anti-Venus immunofluorescence of Neuro2A cells showing concentration of endogenous PDZD8 (tagged with Venus) around Lamp1-positive vesicles and an additional diffuse punctate fluorescence as expected for an ER protein in fixed cells. (Scale bars, 3 µm.)
Fig. 2.
Fig. 2.
PDZD8-mediated ER-wrapping of late endosomes/lysosomes is dynamic and correlates with presence of Rab7. (A) Confocal time-lapse images of PDZD8-EGFP and mCherry-Rab7a showing progressive ER wrapping and PDZD8 enrichment around the vesicles that acquire mCherry-Rab7a. (B) Confocal time-lapse images of Cos7 cells not overexpressing Rab7, showing reversible association of PDZD8-EGFP with a Lamp1-RFP–positive vesicle (arrowhead). (Scale bars, 3 µm.)
Fig. 3.
Fig. 3.
The interactions of the C-terminal region of PDZD8 with GTP-bound Rab7 mediates ER tethering to late endosome/lysosomes. (A) Confocal images of Cos7 cells showing colocalization between PDZD8-EGFP and constitutively active Rab7 (mCherry-Rab7Q67L), but not with its dominant negative Rab7 (mCherry-Rab7T22N). (Scale bars, 3 µm.) (B–E) Representative electron micrographs of Cos7 cells overexpressing PDZD8-EGFP and mCherry-Rab7Q67L. (Scale bars, 150 nm.) Note a vesicle completely surrounded by the ER in the plane of the section in B, while there are no extensive contacts between the ER and mitochondria in the neighboring cell region. Lysosomes and a multivesicular body enwrapped by the ER are visible in C and D and in E, respectively. A portion of the ER-lysosome contact of field C is shown at higher magnification at right, where arrowheads point to protein densities that likely represent PDZD8-Rab7 tethers between the 2 organelles. (F) Schematic representation of the MCS shown in the images of (B–E). (G and H) GST pull-downs showing enrichment of PDZD8 on GMP-PMP-Rab7 relative to controls in cell lysates (G) and purified PDZD8 (H). (I) Confocal images showing no colocalization of mCherry-Rab7Q67L with PDZD8(1-953)-EGFP, but a striking colocalization with PDZD8(951-1154). (Scale bars, 3 µm.)
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