Rab3 mediates a pathway for endocytic sorting and plasma membrane recycling of ordered microdomains

Abstract

The composition of the plasma membrane (PM) must be tightly controlled despite constant, rapid endocytosis, which requires active, selective recycling of endocytosed membrane components. For many proteins, the mechanisms, pathways, and determinants of this PM recycling remain unknown. We report that association with ordered, lipid-driven membrane microdomains (known as rafts) is sufficient for PM localization of a subset of transmembrane proteins and that abrogation of raft association disrupts their trafficking and leads to degradation in lysosomes. Using orthogonal, genetically encoded probes with tunable raft partitioning, we screened for the trafficking machinery required for efficient recycling of engineered microdomain-associated cargo from endosomes to the PM. Using this screen, we identified the Rab3 family as an important mediator of PM localization of microdomain-associated proteins. Disruption of Rab3 reduced PM localization of raft probes and led to their accumulation in Rab7-positive endosomes, suggesting inefficient recycling. Abrogation of Rab3 function also mislocalized the endogenous raft-associated protein Linker for Activation of T cells (LAT), leading to its intracellular accumulation and reduced T cell activation. These findings reveal a key role for lipid-driven microdomains in endocytic traffic and suggest Rab3 as a mediator of microdomain recycling and PM composition.

Keywords: Rab3; Rab7; endosome; microdomain; raft.

Fig. 1.

Raft association is a determinant for PM recycling. (A) Schematic of TMD constructs from Rosetta predictions. (B) Representative confocal images of GPMVs isolated from cells expressing the RFP-tagged TMD of LAT (raft-TMD) or all-Leu TMD (nonraft-TMD). (Left) TMD fluorescence (magenta), (Middle) an unsaturated lipid marker F-DiO (green) to visualize the nonraft phase, and (Right) merge. Last column is a normalized line scan of protein intensity along the black arrow in merged image showing the two peaks corresponding to raft and nonraft phase intensity, respectively. Background-subtracted ratios of these intensities yield raft partition coefficient, K_p,raft. (C) K_p,raft for various TMDs; larger values reflect higher raft preference. Symbols are individual K_p,raft measurements representative of >3 independent experiments. (D) Confocal images show that TMD constructs with high raft partitioning (Left column) enrich at the PM, while TMDs with low raft partitioning (Right column) were predominantly in intracellular puncta. Insets show cell outlines. (E) Steady-state PM fraction for TMD constructs is strongly related to raft partitioning (quantification details in SI Appendix, Fig. S1). (F) Wide-field microscopy after immunofluorescent labeling of organelle markers (recycling endosome, Rab11; early endosome, Rab5; and lysosome, LAMP1). In merge, black corresponds to colocalization; Inset is 5× zoom of the boxed region. Numbers correspond to mean Pearson colocalization (data in SI Appendix, Fig. S3). Nonraft-TMD probe (all-Leu) accumulates in lysosomes. (G) Cells expressing raft-TMD were treated with 25 μM myriocin and 5 μM zaragozic acid for 48 h prior to quantification of PM fraction. PM localization of raft-TMD is specifically disrupted by inhibiting raft lipid synthesis. Symbols represent the mean ± SD of three individual experiments (10 to 15 cells per condition; exemplary dataset shown in SI Appendix, Fig. S2). (H) Trafficking out of the ER was synchronized for raft-TMD and nonraft-TMD using the RUSH system. Both constructs reach the PM within 2 h after release from the ER. Thereafter, raft-TMD accumulates at the PM, while nonraft-TMD accumulates in endosomes. (I) Wide-field imaging showing disruption of endosomal progression by treatment with 10 μg/mL brefeldin A (BFA) or 50 μM bafilomycin A1 (BafA1) for 2 h results in PM accumulation of both raft and nonraft-TMDs. (J) Western blotting shows that nonraft-TMD is degraded significantly more after 24 h of 50 μg/mL cycloheximide treatment to inhibit new synthesis; points represent independent experiments. (K) Model of putative trafficking routes of raft and nonraft-TMDs that lack other sorting signals. Both progress through the secretory system to reach the PM. After internalization, raft-TMDs are recycled back to the PM via the pathway characterized below; nonraft-TMDs proceed through endosomal maturation to lysosomes. (All scale bars, 10 µm.) Significances in G and J show unpaired t tests; *P < 0.05, and ns > 0.05.

Fig. 2.

Raft-TMD transits the late endosome and requires functional Rab7 for recycling. (A) Cells expressing raft-TMD were transfected with GFP-tagged mutants of various Rabs. Confocal imaging shows transfection with Rab5 (Q79L) and Rab7 (Q67L) but not Rab4 (Q67L) or Rab11 (Q70L), which led to accumulation of raft-TMD in intracellular vesicles. In merge, black corresponds to colocalization between raft-TMD and GFP-Rab mutants. Inset numbers represent mean Pearson colocalization for >10 cells; detailed data in SI Appendix, Fig. S6. (B) Quantification of raft-TMD PM fraction in cells coexpressing the indicated mutant Rab; data points represent independent experiments (10 to 30 cells/experiment); also shown are mean +/− SD of all independent experiments. (C) Occasional raft-TMD puncta colocalized (black in merge) with endogenous Rab7-positive endosomes visualized by immunofluorescence and confocal microscopy. (D) Rab7 knockdown by siRNA resulted in accumulation of raft-TMD in intracellular vesicles. (E) Model of raft-TMD recycling occurring in part from late endosomes. Scale bars in A, C, and D, 10 µm, 2 µm in C and D zoom-ins, and 1 µm in E Inset. (Scale bar in E, 0.5 µm.) Significance in B is one-way ANOVA comparing each Rab mutant to EGFP-transfected control; *P < 0.05.

Fig. 3.

Identification of machinery for raft-mediated recycling. (A) Experimental workflow for identification of effectors of raft-mediated trafficking. (B) Volcano plot of the 156 unique screened proteins. The x axis is the % change of intracellular accumulation of raft-TMD; the y axis is −log (p) from three independent experiments. Hits specific for raft-TMD shown in orange; nonspecific hits shown in yellow. (C) Representative images of knockdown of selected hits in cells stably expressing raft-TMD. Bottom row is a 4X zoom of the area marked by the outlined square. (D) Quantification of raft-TMD PM fraction (>100 cells per siRNA per experiment) repeated three times with colors showing individual cells from independent experiments (open symbols show means of individual experiments). (Scale bar in C, 10 μm.) **P < 0.01, ***P < 0.001 from one-way ANOVA compared to NT siRNA.

Fig. 4.

Rab3 mediates raft protein recycling from endosomes. (A) Confocal images of HEK cells expressing raft-TMD treated with siRNA knockdown of Rab3 (or nontargeting siRNA). Rab3-4KD led to accumulation of raft-TMD in intracellular puncta. (B) Quantification of PM fraction of TMD probes by automated image processing of confocal images (Materials and Methods). Symbols represent individual cells from a representative experiment. (C) Confocal images and (D) resulting quantification of PM fraction of raft-TMD or nonraft-TMD in HeLa cells expressing CRISPRi targeted to Rab3 or nontargeting guide. Rab3 knockdown led to accumulation of raft-TMD in intracellular puncta, with no notable effect on nonraft-TMD. (E) Confocal images of Rab3 CRISPRi cells expressing raft-TMD and either WT or C218A-Rab3A. Transient reexpression of Rab3A rescues raft-TMD PM localization, while the putative palmitoylation mutant Rab3A-C218A does not. (F) Quantification of raft-TMD PM fraction with doxycycline to induce Rab3 KD and/or reexpression of Rab3A variants. (G) Intracellular puncta of raft-TMD resulting from Rab3 knockdown colocalize with endogenous Rab7. Inset numbers are mean colocalization; detailed data are shown in SI Appendix, Fig. S13. (H) Confocal, deconvolved images of Rab3 CRISPRi cells transiently expressing raft-TMD-RFP, GFP-Rab3A, and BFP-Rab7 show colocalization of raft-TMD with Rab7 and Rab3 (verified via z-stack in SI Appendix, Fig. S15). (I) Western blot of density gradient after lysis with Triton X-100 to produce detergent-resistant membranes (DRMs), which contain raft-associated material. DRMs are present in the low-density, top fractions (1 to 4). Quantification of protein present in each fraction of the density gradient. Rab3A is found in DRMs together with raft-TMD and the raft marker Fyn. Nonraft-TMD is excluded from DRMs, as are Rab4 and Rab11. (J) Western blot of an acyl-biotin exchange preparation to detect palmitoylated proteins. Only Rab3A among those tested was robustly palmitoylated. (K) Mutation of the putative palmitoylation site C218A significantly reduces the DRM residence of Rab3A (representative western blot shown in SI Appendix, Fig. S18). (L) Schematic model of “hybrid” endosomes containing Rab3 and Rab7 domains and sorting raft-TMD. Significance in B, D, and F calculated by t test of one experiment representative of three independent repeats; >25 cells/experiment were measured. (Scale bars, A, C, E, and G are 10 µm, zoom in G is 2.5 µm, and H is 1 µm.) *P < 0.05, **P < 0.01, ***P < 0.001, ns > 0.05.

Fig. 5.

Rab3-mediated recycling is essential for T cell activation. (A) Confocal images showing the effect of CRISPRi Rab3 KD on full-length protein localization in HeLa cells. Upon Rab3-KD, a fraction of raft-associated proteins LAT, GFP-GPI, and EGFR redistribute from PM to intracellular puncta. (B) Quantification of Rab3-KD on PM fraction of several full-length transfected proteins. (C) Immunostaining for endogenous LAT and Lck in Jurkat T cells reveals accumulation in intracellular puncta upon shRNA-mediated Rab3-KD. (D) Quantification of shRNA Rab3-KD on endogenous LAT and Lck PM localization. (E) Representative confocal immunofluorescence images of endogenous LAT colocalization with Rab7 (Top) or Rab5 (Bottom) in shRab3-mediated Rab3KD Jurkat T cells. (F) Temporal accumulation of Grb2-mEmerald clusters upon Jurkat activation by OKT3-coated coverslips is reduced by Rab3-KD. (G) TIRF microscopy images of Grb2-mEmerald in Jurkat cells after spreading on OKT3-coated coverslips for 240 s. (H) Translocation of NFAT-GFP in Jurkat cells induced by plating onto OKT3-coated coverslips is abrogated by Rab3-KD. Thin dashed lines are cell outlines; thick solid lines are nuclei identified by DAPI staining. Images are taken 15 min after plating. Inset shows that even 30 min after plating, NFAT is still cytoplasmic in Rab3KD cells. (I) Quantification of NFAT-GFP intensity in the nucleus relative to total cell. Symbols in B, D, and I are means ± SD of three independent experiments with >20 cells/experiment; significances are one-way ANOVA compared to controls. Significance in F is mixed-effects analysis. *P < 0.05, **P < 0.01, and ***P < 0.001. (Scale bars in A, G, and H, 10 µm, and C and E, 5 µm.)