Identification of cytoskeleton-associated proteins in isolated rat liver endosomes

Abstract

The polypeptides of three highly purified endosomal fractions isolated from the livers of oestradiol-treated rats were analysed by Western blotting, and the amount and distribution of intrinsic and cytoskeletal-associated proteins were quantified and studied. The ‘late’ endosomes [multivesicular bodies (MVBs)] had the lowest content of cytoskeletal-associated proteins, the most significant being the presence of 25% of the total dynein found in endosomes. The ‘early’ endosome [compartment of uncoupling receptors and ligands (CURL)] fraction contained kinesin (40% of the total in endosomes), dynein (23%), actin (15%) and tubulin (10%). The receptor-recycling compartment (RRC), also demonstrated to be involved in transcytosis, contained the largest number and enrichment of cytoskeletal proteins: actin (84% of the total in endosomes), α-actinin (90%), dynein (52%), tubulin (91%) and kinesin (45%). We also analysed and compared the presence of different endosomal markers such as Rab4, Rab5 and cellubrevin (vesicle soluble NSF attachment protein receptor) in CURL (41%, 15% and 60%) and in RRC (44%, 75% and 30% respectively). Finally, the expression of annexins I, II, IV and VI was studied: annexin I was equally distributed between MVBs and CURL; annexin II was highly enriched in RRC (95%), annexin IV was equally distributed between CURL and RRC, and annexin VI was enriched in CURL (57%). The results indicate that isolated rat liver endosomes contain all the required molecular machinery for the achievement of their role in intracellular trafficking.