Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway

Rab GTPases are associated with distinct cellular compartments and function as specific regulators of intracellular transport. In the endocytic pathway, it is well documented that Rab5 regulates transport from plasma membrane to early (sorting) endosomes. In contrast, little is known about the precise localization and function of Rab4 and Rab11, which are believed to control endocytic recycling. In the present study we have analysed the protein composition of Rab5- and Rab11-carrying endosomes to gain further insight into the compartmental organization of the endocytic and recycling pathway. Endosome populations of this transport route were purified by immunoadsorption from endosome-enriched subcellular fractions using antibodies directed against the cytoplasmic tail of the transferrin receptor, Rab5 or Rab11. Endocytosed transferrin moved sequentially through compartments that could be immunoadsorbed with anti-Rab5 and anti-Rab11, consistent with the theory that Rab5 and Rab11 localise to sorting and recycling endosomes, respectively. These compartments exhibited morphological differences, as determined by electron microscopy. Although their overall protein compositions were very similar, some proteins were found to be selectively enriched. While Rab4 was present on all endosome populations, Rab5 and Rab11 were strikingly segregated. Furthermore, the Rab11-positive endosomes were rich in annexin II, actin and the t-SNARE syntaxin 13, compared to Rab5-containing endosomes. In an in vitro assay, the Rab5 effector protein EEA1 was preferentially recruited by Rab5-positive endosomes. Taken together, our data suggest an organization of the transferrin pathway into distinct Rab5- and Rab11-positive compartments.