Abstract
Rab15 is a novel endocytic Rab that counters the stimulatory effect of Rab5-GTP on early endocytic trafficking. Rab15 may interfere with Rab5 function directly by sequestering Rab5 effectors or indirectly through novel sets of effector interactions. To distinguish between these possibilities, we examined the effector binding properties of Rab15. Rab15 does not interact directly with the Rab5 effectors rabex-5 and rabaptin-5 in a yeast two-hybrid binding assay. Rather mammalian suppressor of Sec4 (Mss4) was identified as a binding partner for Rab15. Mss4 preferentially binds GDP-bound (T22N) and nucleotide-free (N121I) Rab15, consistent with the proposed role of Mss4 as a chaperone that stabilizes target Rabs in their nucleotide-free form. Mutational analysis of Rab15 indicates that lysine at position 48 (K48Q) is important for the binding of Rab15-GDP to Mss4. Moreover, the mutation K48Q counters the inhibitory phenotype of wild type Rab15 on receptor-mediated endocytosis in HeLa cells and homotypic endosome fusion in vitro without altering the relative amount of cell surface-associated transferrin receptor. Together, these data indicate a novel role for Mss4 as an effector for Rab15 in early endocytic trafficking.
Highlights
Endocytosis of cell surface receptors regulates both the intensity and duration of receptor signaling by controlling the location of signaling interactions and the desensitization and recycling of activated receptors
Does Rab15 Bind Rab5 Effectors?—Given the opposing effects of Rab15 and Rab5 on early endocytosis, it is highly likely that the transport steps regulated by these Rabs overlap at some point within the endocytic network, possibly in terms of a common effector or target
Rabaptin-5 interacts with Rab4 through a distinct binding site [25] suggesting a role for this effector in recycling from early endosomes to the cell surface
Summary
Endocytosis of cell surface receptors regulates both the intensity and duration of receptor signaling by controlling the location of signaling interactions and the desensitization and recycling of activated receptors (reviewed in Refs. 1 and 2). A model is emerging in which Rab5GTP functions as a regulatory protein, driving assembly of specific effector complexes on endosomal membranes leading to membrane fusion [24] Consistent with this model, the early endocytic GTPases, Rabs 4 and 11, have been shown to organize into distinct domains on early endosomes through the local recruitment of effectors [14, 15]. Overexpression of activated Rab (Rab15-GTP) inhibits both fluid phase and receptor-mediated endocytosis in vivo and the homotypic fusion of early endosomes in vitro. Mutations that constitutively inactivate Rab (Rab15-GDP) stimulate early endocytosis and fusion of homotypic endosomes in vitro [27] These data suggest that Rab functions to reduce endocytic trafficking, primarily at the level of early/sorting endosomes. Given the opposing effects of Rab and Rab on early endocytosis, the transport steps regulated by these GTPases likely intersect at some point within the endocytic network, presumably through the action of a shared effector or accessory
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