Fig. 8. The effect of Rab22a on the transport of aspartylglucosaminidase (AGA) to lysosomes. BHK-21 cells were double transfected for 24 hours with Rab22a wt or mutant cDNAs (indicated on the left) in pcDNA3.1 or the plain vector plasmid (Mock) and human AGA cDNA in pSVPoly, followed by a 3 hour chase in the presence of cycloheximide (25 µg/ml). Thereafter the cells were triple immunostained for AGA, EEA1 and LBPA, and viewed under a confocal microscope. Staining for AGA is shown in panels A,F,K,P, for EEA1 in B,G,L,Q, and for LBPA in C,H,M,R. Pairwise overlays of the channels are shown in D,E,I,J,N,O,S and T. In mock-transfected cells (A-E), the AGA was chased into punctate structures that colocalized with the late endosome/lysosome marker LBPA but not with EEA1. In a majority of cells expressing the wt Rab22a, the trafficking of AGA to late endocytic compartments proceeded in a similar fashion, despite the presence of enlarged EEA1-positive endosomes induced by the GTPase (F-J). In cells expressing the Q64L mutant, however, AGA accumulated in large vacuolar endosomes, which contained both early (EEA1) and late (LBPA) endosomal markers (K-O). Expression of the Rab22a S19N mutant had no significant effect on the transport of AGA to late endocytic compartments (P-T). Bar=10 µm.

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