Figure 3.

Suppression of p150^glued Expression Perturbs the Efficiency of Retromer-Mediated Endosome-to-TGN Transport

(A) Demonstration of knock down of p150^glued in HeLa cells using three different siRNAs and probing for endogenous p150^glued, using tubulin staining to indicate equal loading.

(B) Quantification of knock down obtained in four different experiments for p150^glued suppression using three different siRNAs each. Error bars indicate standard deviation.

(C) Suppression of p150^glued affects the steady-state distribution of CI-MPR in HeLa cells, leading to a peripheral dispersal of the label in comparison to control cells, phenocopying loss of retromer components. Note that under these conditions, knock down of p150^glued does not affect the general organization of the giantin-stained Golgi, the TGN, the microtubule array, or EEA1-positive early endosomes, while a minor alteration in the distribution of the CD63-positive late endosomal/lysosomal compartment is apparent (Figure S4).

(D) Quantification of the number of Golgis with more than half-maximal intensity of CI-MPR staining expressed as percent of total Golgis analyzed, using three different siRNAs for p150^glued suppression and including SNX1 knock down as a positive control for defective retromer trafficking. Error bars indicate standard deviation.

(E) Kinetics of the uptake of a CD8-tagged CI-MPR from the plasma membrane and trafficking to the TGN, quantified as percent of CD8 label in the TGN. Knock down of p150^glued or SNX1 induces a major reduction in the efficiency of transport to the TGN in comparison to control cells. Error bars indicate standard deviation.