Fig. 4: The cell surface proteome is re-modelled in VPS35 KO H4 cells.

a Scatter plot of VPS35 KO/VPS35-GFP transmembrane protein abundances in the cell surface proteome (x-axis) versus the LysoIP proteome (y-axis). Datapoints coloured based on p-value scores in each experiment (green—significantly altered in LysoIP, magenta—significantly altered in surface proteome, blue—significantly altered in LysoIP and surface proteome), derived from two-tailed paired t tests. b Heatmap depicting the loss of transmembrane proteins from the cell surface proteome (purple columns) with concomitant enrichment in the LysoIP proteome (green columns) in VPS35 KO cells relative to wild-type and VPS35-GFP rescue controls. Proteins coloured by Log₂ fold change between VPS35 KO and wild-type (WT) or VPS35-GFP (rescue) samples. c Cell surface and lysosomal proteomes were subjected to immuno-blotting with antibodies recognising defined Retromer cargoes (ATP7a and GLUT1). Data representative of 6 (cell surface) and 7 (LysoIP) independent repeats. kDa = kilodaltons. d Heatmap depicting the enrichment of transmembrane proteins in the cell surface proteome (purple columns) and corresponding abundance in the LysoIP proteome (green columns) in VPS35 KO cells relative to wild-type and VPS35-GFP rescue controls. Proteins coloured by Log₂ fold change between VPS35 KO and wild-type (WT) or VPS35-GFP (rescue) samples. e Analysis of significantly enriched pathways in the VPS35 KO transmembrane cell surface proteome relative to wild-type and VPS35-GFP rescue controls, hypergeometric test. f Volcano plot of VPS35 KO/VPS35-GFP protein abundances in the growth media ‘secretome’. 325 proteins were significantly enriched in the growth media of VPS35 KO cells compared to both wild-type and rescue sample (Log₂ fold change > 1, p < 0.05), two-tailed paired t tests. Magenta—significantly depleted in VPS35 KO, green—significantly enriched in VPS35 KO. g Gene ontology analysis of cellular components enriched in the VPS35 KO ‘secretome’. Statistically significant categories (p < 0.05) displayed in green, Fisher’s exact test. h Dot-plot predicting lysosomal luminal protein and APP processing protein abundances in the VPS35 KO ‘secretome’ relative to wild-type and VPS35-GFP rescue control samples. Log₂ fold change, relative change and p-value score are depicted by dot colour, size and outline respectively, two-tailed paired t tests.