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. 2013 Jan 1;126(Pt 1):45-52.
doi: 10.1242/jcs.112904. Epub 2012 Sep 26.

Clathrin is not required for SNX-BAR-retromer-mediated carrier formation

Ian J McGough  1 , Peter J Cullen
Affiliations

Clathrin is not required for SNX-BAR-retromer-mediated carrier formation

Ian J McGough et al. J Cell Sci. .

Abstract

Clathrin has been implicated in retromer-mediated trafficking, but its precise function remains elusive. Given the importance of retromers for efficient endosomal sorting, we have sought to clarify the relationship between clathrin and the SNX-BAR retromer. We find that the retromer SNX-BARs do not interact directly or indirectly with clathrin. In addition, we observe that SNX-BAR-retromer tubules and carriers are not clathrin coated. Furthermore, perturbing clathrin function, by overexpressing a dominant-negative clathrin or through suppression of clathrin expression, has no detectable effect on the frequency of SNX-BAR-retromer tubulation. We propose that SNX-BAR-retromer-mediated membrane deformation and carrier formation does not require clathrin, and hence the role of clathrin in SNX-BAR-retromer function would appear to lie in pre-SNX-BAR-retromer cargo sorting.

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Figures

Fig. 1.
Fig. 1.
Retromer SNX-BARs do not associate with CHC. (A) Retromer SNX-BARs do not colocalise with CHC. HeLa cells were fixed and stained for endogenous SNX1 or SNX6 (green) and endogenous CHC or VPS35 (red) or virally transduced with GFP-SNX2 or GFP-SNX5 (green), or transiently transfected with GFP-OCRL1, prior to being fixed and stained for endogenous CHC (red). Scale bars: 10 µm. (B) SNX-BAR-retromer-labelled endosomes lie juxtaposed to clathrin-labelled punctae. HeLa cells transiently expressing CLC–dsRed were fixed and stained for endogenous SNX1. Scale bar: 1 µm. (C) Analysis of the colocalisation shown in A: the percentage overlap of the individual SNX–BAR or OCRL1 signals with the clathrin signal, and the degree of correlation when signals overlap (Pearson’s correlation coefficient). Error bars show the s.d. (n = 3 with 20 cells per condition). (D) Sequence alignment of the retromer SNX-BARs reveals the conserved putative clathrin-binding box (−)ΦpΦL [L, leucine; Φ, bulky hydrophobic; p, polar; (−), negative]. The amino acids fused to GFP to create isolated clathrin-binding boxes used in E are shaded pink. (E) Isolated clathrin-binding boxes fail to bind clathrin. Extracts from HEK293 cells transiently expressing GFP-tagged clathrin-binding boxes from SNX1, SNX2, SNX5 and SNX6 and full-length SNX1, SNX2, SNX5, SNX6 and SNX15, were subjected to a GFP-nanotrap prior to western blotting for endogenous CHC and RME-8. (F) Putative clathrin-binding boxes are predicted not to be accessible when the SNX-BARs are bound to PtdIns3P-enriched endosomal membranes. NMR structures of SNX1 and SNX5 are shown. Key residues required for PtdIns3P binding are shown in green (SNX1: Arg186 and Arg238; SNX5: Arg42, Lys44, Lys46, Lys96 and Arg103), whereas the clathrin-binding boxes are shown in red. (G) Full-length SNX1, SNX2 or SNX5 do not directly bind the terminal domain of CHC. Glutathione resin containing the correctly folded clathrin or a denatured clathrin (boiled for 20 minutes) were incubated with purified 2 µM SNX1, SNX2 or SNX5. SNX15 or SNX15 without its clathrin-binding box were used as positive and negative controls. (H) Clathrin is not detected in SNX-BAR-retromer immunoprecipitates. Cell extracts derived from HEK293 cells transiently transfected with GFP, GFP-SNX1, GFP-SNX2, GFP-SNX5, GFP-SNX6, GFP-VPS35 and GFP-SNX15, were subjected to GFP-nanotrap analysis and blotted for CHC and GFP.
Fig. 2.
Fig. 2.
Clathrin is absent from SNX1-retromer tubules and carriers. RPE-1 cells were transiently co-transfected with pEGFP-SNX1 (green) and CLC-dsRed (red) and imaged live after a 16 hour incubation period. (A,B) Frames depicting the formation and scission of a GFP–SNX1 tubule from a vesicle positive for both SNX1 and clathrin (A), or positive for SNX1 but negative for clathrin (B; in both cases the arrowheads indicate the dual-expressing vesicle, whereas the arrow indicates the carrier after scission; supplementary material Movies 1, 2). Scale bars: 4 µm. (C) Of 77 SNX1-decorated tubules all were clathrin negative, whereas of 77 tubulating endosomes, 22 were clathrin positive. (D) SNX1 colocalises with RME-8. HeLa cells were fixed and stained for endogenous SNX1 (green) and endogenous RME-8 (red). Arrows indicate endosomes positive for both. Scale bar: 10 µm. (E) RME-8 localises to SNX1-positive, clathrin-negative sub-domains. HeLa cells transiently expressing CLC–dsRed were fixed and stained for endogenous SNX1 (blue) and endogenous RME-8 (green). Scale bars: 1 µm. (F) SNX1 tubules emanate from SNX1- and RME-8-positive sub-domains. HeLa cells transiently expressing CLC-dsRed were fixed and stained for endogenous SNX1 (blue) and endogenous RME-8 (green). Arrow indicates a tubulating endosome. Scale bar: 1 µm. (G) Similar to SNX1, VPS35 is juxtaposed to clathrin, in a domain positive for CI-MPR. HeLa cells transiently expressing CLC-dsRed were fixed and stained for endogenous VPS35 (blue) and endogenous CI-MPR (green). Scale bars: 1 µm.
Fig. 3.
Fig. 3.
Clathrin is not required for SNX-BAR-retromer tubulation. (A) Overexpression of the terminal domain of CHC does not affect SNX-BAR-retromer tubulation. HeLa cells were transiently transfected with a CDM8–T7–CHC-hub domain construct or a empty CDM8 construct. Cells were incubated in serum-free medium for 1 hour prior to addition of serum-free medium containing 25 µg/ml of fluorescently labelled transferrin and then fixed after 30 minutes and stained for endogenous SNX1 (green) and T7 (red) after a 24 hour incubation period. Scale bar: 10 µm. Examples of SNX1 tubules are indicated with arrows in the enlarged image i (scale bar: 3 µm). (B) Number of SNX1 tubules per cell (n = 3 and 10 cells per condition). (C) RNAi-mediated suppression of CHC does not affect SNX-BAR-retromer tubulation. CHC was knocked down in HeLa cells. Coverslips were incubated in serum-free medium for 1 hour prior to addition of serum-free medium containing 25 µg/ml of fluorescently labelled transferrin and then fixed after 20 minutes. Cells were subsequently stained for endogenous SNX1 and CHC, to identify efficient knockdown. Arrows indicate SNX1 tubules. The regions within the dashed lines are enlarged in the panels to the right. Scale bars: 10 µm; 1 µm in enlarged images.
Fig. 4.
Fig. 4.
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