Rab Effector Protein Research Articles

IL4/PGE 2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E 2 (PGE 2) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.

The Rab-interacting lysosomal protein, a Rab7 and Rab34 effector, is capable of self-interaction

Rab-interacting lysosomal protein (RILP) has been identified as an interacting partner of the small GTPases Rab7 and Rab34. Active Rab7 recruits RILP on the late endosomal/lysosomal membrane and RILP then functions as a Rab7 effector controlling transport to degradative compartments. Indeed, RILP induces recruitment of dynein–dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Recently, Rab7 and RILP have been found to be key proteins also for the biogenesis of phagolysosomes. Therefore, RILP represents probably an important factor for all endocytic routes to lysosomes. In this study, we show, using the yeast two-hybrid system, that RILP is able to interact with itself. The data obtained with the two-hybrid system were confirmed using co-immunoprecipitation in HeLa cells. The data together indicate that RILP, as already demonstrated for several other Rab effector proteins, is capable of self-association, thus probably forming a homo-dimer.

Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway.

Synthetic peptides of the putative effector domain of members of the ras-related rab gene family of small GTP-binding proteins were synthesized and found to be potent inhibitors of endoplasmic reticulum (ER) to Golgi and intra-Golgi transport in vitro. Inhibition of transport by one of the effector domain peptides was rapid (t1/2 of 30 s), and irreversible. Analysis of the temporal site of peptide inhibition indicated that a late step in transport was blocked, coincident with a Ca2(+)-dependent prefusion step. The results provide novel biochemical evidence for the role of members of the rab gene family in vesicular transport in mammalian cells, and implicate a role for a new downstream Rab effector protein (REP) regulating vesicle fusion.