SNX2 is capable of sensing membrane curvature but does not induce membrane tubulation in vitro or in vivo. (A) Variously curved liposomes were formed by extrusion, and binding of proteins was assayed by sedimentation. SNX2 bound preferentially to highly curved membranes, as did SNX1. The epsin1 ENTH domain (which strongly tubulates membranes), and Dab2 (which does not change liposome shape) were insensitive to curvature. Amount of bound protein was normalised to the value for 0.8 μm liposomes. Average values for three experiments ± s.d. are shown. (B) Liposomes derived from brain were incubated with 20 μM BSA (i) or 20 μM of full length recombinant SNX1 (ii,iii) or SNX2 (iv,v). Whereas budding tubular profiles and occasionally tubular networks were observed with SNX1, no tubules were observed when using SNX2. For SNX1 (ii,iii) and SNX2 (iv,v) the data represents two independent experiments. (C). HeLa cells were transiently transfected with a construct encoding for GFP-SNX2. After 48 hours of incubation, cells expressing low and high levels of GFP-SNX2 were fixed and imaged by confocal microscopy. At low levels, SNX2 was associated with punctate cytoplasmic vesicles, which contrasted with the swollen endosomal vacuoles observed under conditions of high level expression. Bars, 10 μm.

At low-level expression, GFP-SNX2 is associated with elements of the early endosome. HeLa cells were transiently transfected with GFP-SNX2 and, after 24 hours, cells were fixed and stained for the early endosomal markers EEA1 (A) and SNX1 (B), internalised receptors for transferrin (C) and EGF (D), and the late endosomal marker LAMP1 (E). Images are representative of more than 10 imaged cells in each case. Bars, 10 μm.

In HeLa cells endogenous SNX2 and SNX1 co-localise on an early endosomal compartment. (A) HeLa cells were treated with control, SNX2-specific, SNX1-specific or both SNX2 and SNX1 siRNA duplexes for 72 hours. At this time, the levels of SNX2 and SNX1 were determined by western blotting with SNX2 or SNX1 antisera. In all cases loading was controlled by probing samples with an anti-tubulin antibody. Quantification of the data revealed that, for individual siRNA treatment using SNX2-specific or SNX1-specific duplexes, the level of SNX2 and SNX1 expression was suppressed by 82% and 93%, respectively. Under conditions of simultaneous duplex treatment, SNX2 and SNX1 were suppressed by 81% and 92%, respectively. (B) HeLa cells were treated with control, SNX2-specific, SNX1-specific or both SNX2 and SNX1 siRNA duplexes for 72 hours prior to fixation. Cells were stained for endogenous SNX1 and SNX2. Bar, 10 μm. (C) HeLa cells were fixed and co-stained with SNX1- and SNX2-specific antibodies to reveal the endogenous distribution of these proteins. Bar, 10 μm.

Endogenous SNX2 and SNX1 co-localise on tubular elements of the early endosome. Ultrathin cryosections of HepG2 cells showing the co-localization of SNX2 with SNX1 and CI-MPR on endosomal tubules. SNX2 (15 nm gold particles) and SNX1 (10 nm gold particles) co-localize on tubules emanating from endosomal (E) vacuoles (arrow in A) and on numerous vesicular-tubular profiles in close vicinity of endosomal vacuoles (arrows in B). SNX2 (15 nm gold particles) associates with CI-MPR (10 nm gold particles)-positive endosomal buds (arrows in C) and similar endosome-associated vesicular-tubular profiles, as shown in B (arrow in D). Bars, 200 nm

Suppression of SNX2 or the joint suppression of SNX2 and SNX1 has no gross affect on EGF or transferrin receptor sorting. (A) HeLa cells were treated with either control, SNX2-specific siRNA or jointly with SNX1- and SNX2-specific siRNA duplexes for 72 hours. Cells were serum-starved for 3 hours and labelled with 1 kBq per well ¹²⁵I-EGF for 1 hour at 4°C, allowed to internalise surface bound ¹²⁵I-EGF for 5 minutes at 37°C, and then returned to 4°C. Cells were chased into 100 ng/ml cold EGF-containing media for various times at 37°C. Recycled, degraded and internalised fractions were subjected to gamma counting. Data is the average±s.d. from three independent experiments. (B) HeLa cells were treated with control, SNX2-specific siRNA or jointly with SNX1- and SNX2-specific siRNA duplexes for 72 hours. Cells were serum-starved for 3 hours and labelled with 1 kBq per well ¹²⁵I-transferrin for 60 minutes at 37°C. Cells were chased into 50 μg/ml cold transferrin-containing media for various times at 37°C. Recycled, degraded and internalised fractions were subjected to gamma counting. Data is the average±s.d. from three independent experiments. (C) Western analysis of a typical SNX1 and SNX2 suppression achieved during one of the three sets of receptor trafficking assays.

Endogenous SNX2 shows a high degree of co-localisation with the retromer component mVps26. (A) Co-localisation of endogenous SNX2 and endogenous mVps26. Cells were plated on coverslips, fixed and stained for SNX2 and mVps26. (B) Suppression of SNX1, SNX2, or joint suppression of both SNX1 and SNX2 does not affect mVps26 localisation. HeLa cells were treated with control, SNX1-specific siRNA, SNX2-specific siRNA or jointly with SNX1- and SNX2-specific siRNA duplexes for 72 hours. Cells were then fixed and stained against endogenous SNX1, endogenous SNX2 and endogenous mVps26. Individual or joint suppression of SNX1 and SNX2 does not affect punctate distribution of mVps26. Bars, 20 μm (A,B).

Suppression of SNX2 does not result in a detectable defect in the steady-state distribution of the CI-MPR. (A) HeLa cells were treated with control, SNX1-specific siRNA, SNX2-specific siRNA or jointly with SNX1- and SNX2-specific siRNA duplexes for 72 hours. Cells were fixed and stained against endogenous SNX1, endogenous SNX2 and endogenous CI-MPR. In cells subjected to RNAi against SNX2, the CI-MPR remains at a perinuclear structure that has been previously characterised as the TGN. In cells in which SNX1 and SNX2 have been suppressed, the CI-MPR undergoes a limited redistribution at steady state to peripheral structures. Bars, 20 μm. (B) HeLa cells were treated twice with control, SNX1-specific siRNA, SNX2-specific siRNA or jointly with SNX1- and SNX2-specific siRNA duplexes for two consecutive periods of 72 hours. At this time, cells were washed into cycloheximide (40 μg/ml)-containing media for 0, 4, 8 or 12 hours. Cells were lysed and the levels of endogenous CI-MPR present were resolved by western blotting with CI-MPR-specific antisera, followed by volume integration. Results are presented graphically from the averages of four independent experiments.