Rab32 interacts with SNX6 and affects retromer-dependent Golgi trafficking

Abstract

The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson's disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2.

Fig 1. Rab32 interacts with the PX domain of SNX6.

Rab32-interacting domains were identified by constructing different plasmids containing either full length SNX6 or the BAR (SNX6ΔN) or PX (SNX6ΔC) domains only. Co-transformations were peformed in the yeast strain Gold with the indicated plasmids. Colony growth on QDO plates and blue color indicates that the proteins interact (+), n≥3 independent experiments.

Fig 2. Rab32, Rab38 and SNX6 binding in co-immunoprecipitation and GST-pulldown experiments.

(A) Lysates from IHKE-1 cells stably expressing GFP-Rab32 were used for co-immunoprecipitation of endogenous SNX6 with the GFP-Trap kit. IHKE-1 cells transiently transfected with GFP alone were used as control. n = 2 independent experiments. (B) 5 μg GST-Rab32 wt, GST-Rab38 wt, GST-Rab1A wt or GST as control were loaded to glutathione agarose beads followed by incubation with IHKE-1 lysate overnight. Samples were analyzed by SDS-PAGE and subsequent Western blot analysis against SNX6. n = 3 independent experiments.

Fig 3. Co-localization analysis of SNX6 with endogenous, constitutively active, inactive and wild-type Rab32.

(A) IHKE-1 cells were cultured on glass cover slips for 24 hours, fixed and subsequently stained with antibodies against Rab32 (cy3, red channel) and SNX6 (cy2, green channel). Blue: DAPI staining of the nucleus. (B) IHKE-1 cells were either transiently transfected with plasmids encoding GFP-Rab32 T39N or stably expressing GFP Rab32^WT or GFP-Rab32 Q85L, fixed and subsequently stained with an antibody against SNX6. GFP = green channel; SNX6 (cy3) = red channel (C) HeLa cells cultured on glass cover slips were transfected with constructs encoding GFP-SNX6 or DsRed-Monomer-Rab32 wt. After 24h cells were fixed and analyzed by flourescece microscopy. GFP = green; DsRed-Monomer = red. Arrows indicate co-localization, scale bar = 10μm.

Fig 4. Rab32 influence on retromer mediated transport of Shiga toxin B.

(A) HeLa cells transfected with either GFP-Rab32 Q85L, GFP-Rab32 T39N (upper row), GFP-Rab6A’ Q72L or GFP-Rab6A’ T27N (lower row). 24 hours later cells were incubated with STxB-cy3 and pulse-chased for 60 minutes, followed by fixation in 4% PFA. Rab32-expressing cells were additionally stained by immunofluorescent labeling of Rab6A. (blue) to have a comparable Golgi marker to the GFP-Rab6A’ transfected cells. The Rab6A/A’ channels were depicted in green, STxB-cy3 in red. STxB co-localizing with the Golgi apparatus appears in yellow. GFP-Rab32 constructs are not visible. Scalebar = 10 μm (B) Quantification of STxB transport rates. Endogenous Rab6A or GFP-Rab6A’ was used to determine the localization of the Golgi apparatus. Transport rate is given as % co-localization of STxB with the Golgi apparatus. A lower % co-localization indicates a reduced rate of STxB transport. Rab32 Q85L: n = 127 cells, Rab32 T39N: n = 119 cells, Rab6A’ Q72L: n = 132 cells; Rab6A’ T27N: n = 131 cells.

Fig 5. Rab32 and SNX6 influence on cation independent mannose-6 phosphate receptor (CI-MPR) trafficking in IHKE-1 cells.

(A) IHKE-1 cells transfected with either GFP-Rab32 T39N, GFP-Rab32^WT or GFP-Rab32 Q85L. After 24 hours cells were fixed in 4% PFA and subsequently stained for CI-MPR. (B) IHKE-1 cells transfected with either GFP-SNX6 or GFP-SNX6ΔC. After 24 hours, cells were fixed in 4% PFA and subsequently stained for CI-MPR. (C) IHKE-1 cells transfected with either GFP-Rab32^WT or GFP-Rab32^WT and myc-SNX6ΔC. After 24 hours cells were fixed in 4% PFA and subsequently stained for CI-MPR. Successful double transfection was assesed by immunofluorence against myc-tag (not shown) demonstrating >95% co-transfected cells. (D) Quantification of CI-MPR distribution in the cell for different Rab32 constructs. MPR-signal was either categorized as dispersed phenotype or not disperesed. n for control, GFP-Rab32^WT, -Q85L or -T39N were 910, 210, 275 and 110 cells from ≥3 independent experiments. (E) Quantification of CI-MPR distribution in the cell for different SNX6 constructs MPR-signal was either categorized as dispersed phenotype or not dispersed. n for control, GFP-SNX6, GFP-SNX6ΔC or Rab32 wt+SNX6ΔC were 910, 54, 57 and 124 cells from ≥2 independent experiments. Significance was determined by Fisher’s Exact Test; p<0.005 = ***, n.s. = not significant; Scalebar = 10 μm.

Fig 6. Co-localization analysis of GFP-LRRK2 with endogenous Rab32 and SNX6.

SH-SY-5Y stably expressing GFP-LRRK2 cells were cultured on glass cover slips for 48 hours. Then, cells were stained with antibodies against Rab32 and SNX6. Secondary antibodies were coupled to cy3 or Alexa 647. Cells were analyzed wit a Zeiss SIM microscope. n = 2 independent experiments. Colors in the merge image: GFP = green, cy3 = red, Alexa 647 = blue color; Scale bar = 10μm.