Sequential action of Rab proteins along the endocytic-recycling pathway

Abstract

The endocytic pathway in eukaryotic cells is organized into distinct compartments. Early endosomes are heterogeneous and dynamic organelles, which are found both spread through the cortical cytoplasm (sorting-early endosomes) and in the perinuclear region (recycling endosomes). Endocytosed molecules that enter sorting endosomes can be directed to the degradative pathway or recycled back to the plasma membrane, either directly or by passing through perinuclear-recycling endosomes. Very little is known about the molecular mechanisms that regulate transport between these different compartments and the functional properties of endocytic organelles. In this thesis, I have investigated the molecular regulation of the endocytic-recycling pathway and, in particular, how endocytosis and recycling are co-ordinated at the level of early endosomes. To this end, I have addressed the function of Rab5, Rab4, and Rabll, three small GTPases that control trafficking from the plasma membrane to the early endosomes, sorting of cargo within early endosomes and recycling to the plasma membrane, respectively. Rab proteins function in their GTP-bound active state through the recruitment of effector proteins to the membrane compartment where they are localized. The identification of a large number of Rab5 effectors, together with the realization that these proteins act co-operatively, led to the proposal that this GTPase organizes the endosomal membrane into a biochemically and functionally distinct membrane domain. If generalized to other family members, this model would predict that other Rab proteins present on endosomes such as Rab4 and Rabll should be localized to discrete, non-overlapping membrane domains distinct from the one occupied by Rab5. Moreover, the finding that one Rab5 effector, Rahaptin-5, interacts also with Rah4 raises the possibility that the activity of these two GTPases is molecularly co-ordinated. Given the complementary role of Rab5 and Rab4 in regulating entry in, and exit out, of early endosomes the coupling of their activity through a common effector may ensure co-ordination of the endocytic and recycling functions of early endosomes. I have experimentally tested the working hypothesis predicting that divalent Rab effectors may regulate the association between contiguous Rab domains to allow the sequential transport of cargo along the endocytic-recycling pathway. To provide evidence for this model, two complementary approaches have been undertaken. First, I have developed an in vitro recycling assay to identify and characterize molecules involved in this process. Second, I have conducted a high-resolution morphological analysis of the endocytic recycling pathway in stable cell lines expressing GFP-tagged versions of Rab proteins. The results obtained support the idea that endosomes are indeed organized into distinct domains, harbouring Rab5, Rab4 and Rabll. The association between these domains follows a non-random distribution giving rise to three major populations of endosomes: one containing Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rabll. Upon endocytosis, recycling cargo first enters Rab5 domains, and then progresses through Rab5/Rab4 and Rab4/Rabll endosomes before returning to the plasma membrane. Based on these observations, I then addressed the question of how the communication between neighbouring domains is regulated by specifically searching for Rab5 and Rab4 common effectors using an affinity chromatography approach. The results of these experiments led me to identify Rahenosyn-5 as a novel Rab5 and Rab4 common effector. Subsequently, I demonstrated that Rabenosyn-5, as well as Rabaptin-5 over-expression specifically increased the association between Rab5 and Rab4 endosomal domains and stimulated transferrin recycling. Concomitantly, the fraction of Rah4+Rabll positive structures was reduced and transport to peri-nuclear recycling endosomes decreased. Thus, divalent Rab5 and Rab4 effectors regulate endocytosis and recycling by connecting Rab5 and Rab4 domains on early endosomes. These results provide support for the hypothesis that Rab proteins and their effectors regulate the compartmental organization and trafficking function of early endosomes.