FREE-FLOW ELECTROPHORETIC ANALYSIS OF ENDOSOME SUBPOPULATIONS OF RAT HEPATOCYTES

The separation of functional early and late endosomes from other cellu lar compartments by free-flow electrophoresis (FFE) has been previousl y demonstrated in nonpolarized cells [1, 2]. Here, using I-125-labeled anti-secretory component antibodies ([I-125]SC Ab) and FITC-labeled a sialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysoso mal pathway, respectively, we demonstrate the separation of three dist inct endosome subpopulations from polarized rat hepatocytes. Internali zation of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytoti c and the lysosomal pathways are arrested in the sorting early endosom e at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 d egrees C, transcytotic endosomes carrying [I-125]SC Ab migrated with a n electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anod e than early endosomes. These data indicate that in rat hepatocytes, t he transcytotic and lysosomal pathways utilize a common (i.e. early en dosomes) and two distinct endosome subpopulations (i.e. transcytotic e ndosomes, late endosomes) prior to delivering proteins for biliary sec retion or lysosomal degradation, respectively.