The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway.

The mechanochemical forces that move and position intracellular organelles and their intermediates in eukaryotic cells are provided by molecular motor proteins which include the cytoplasmic dynein-1 motor complex. Recently, we identified the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) as a novel binding-partner for dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1), a subunit of cytoplasmic dynein-1. Here, we show that FIP3 also binds the dynein light intermediate chain 2 subunit (DLIC-2, gene symbol DYNC1LI2). We show that like DLIC-1, DLIC-2 binds the amino-terminal 435 amino acids of FIP3 and that FIP3 links Rab11a to DLIC-2. We also show that FIP3 recruits DLIC-2 onto membranes and that DLIC-2 is necessary for the accumulation of endocytosed-transferrin (Tfn) at the pericentrosomal endosomal-recycling compartment (ERC). Finally, we demonstrate that overexpression of FIP3 fragments the Golgi complex by sequestering cytoplasmic dynein-1. In conclusion, we have identified FIP3 as the first membrane-associated interacting-partner for DLIC-2 and propose that this interaction serves to control endosomal trafficking from sorting endosomes to the ERC.

Co-ordination of Rab GTPase function has emerged as a crucial mechanism in the control of intracellular trafficking processes in eukaryotic cells. Here, we show that GRAB/Rab3IL1 [guanine nucleotide exchange factor for Rab3A; RAB3A interacting protein (rabin3)-like 1], a protein that has previously be shown to act as a GEF (guanine nucleotide exchange factor) for Rab3a, Rab8a and Rab8b, is also a binding partner for Rab11a and Rab11b, but not the closely related Rab25 GTPase. We demonstrate that exogenous expression of Rab11a and Rab11b shift GRAB's distribution from the cytoplasm onto membranes. We find that the Rab11a/Rab11b-binding region of GRAB lies within its carboxy-terminus, a region distinct from its GEF domain and Rab3a-binding region. Finally, we describe a GRAB deletion mutant (GRABΔ223-228) that is deficient in Rab11-binding ability. These data identify GRAB as a dual Rab-binding protein that could potentially link Rab3 and Rab11 and/or Rab8 and Rab11-mediated intracellular trafficking processes.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface.