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Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.

The Rab11-family interacting protein 3 (Rab11-FIP3), also known as Arfophilin and Eferin, is a Rab11 and ADP-ribosylation factor (ARF) binding protein of unknown function. Here, we sought to investigate the subcellular localisation and elucidate the function of Rab11-FIP3 in eukaryotic membrane trafficking. Utilising a polyclonal antibody specific for Rab11-FIP3, we have demonstrated by immunofluorescence microscopy that Rab11-FIP3 colocalises with Rab11 in a distinctive pericentrosomal location in A431 cells. Additionally, we found that Rab11-FIP3 localises to punctate vesicular structures dispersed throughout A431 cells. We have demonstrated that both Rab11 and Rab11-FIP3 localise to the cleavage furrow during cytokinesis, and that Rab11-FIP3 localisation is dependent on both microtubule and actin filament integrity. We show that Rab11-FIP3 does not enter brefeldin A (BFA) induced membrane tubules that are positive for the transferrin receptor (TfnR). Furthermore, we show that expression of an amino-terminally truncated mutant of Rab11-FIP3 (Rab11-FIP3((244-756))) does not inhibit transferrin (Tfn) recycling in HeLa cells. It is likely that Rab11-FIP3 is involved in trafficking events other than Tfn trafficking; these may include the transport of endosomally derived membrane to the cleavage furrow during cytokinesis.

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.

We have recently identified Rab11-FIP4 as the sixth member of the Rab11-FIP family of Rab11 interacting proteins. Here, we demonstrate that Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and that its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). We investigated the subcellular localisation of Rab11-FIP4 in HeLa cells and show that it colocalises extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. We demonstrate that the carboxy-terminal region of Rab11-FIP4 (Rab11-FIP4(C-ter)) is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling.

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells (Hales, C. M., Griner, R., Hobdy-Henderson, K. C., Dorn, M. C., Hardy, D., Kumar, R., Navarre, J., Chan, E. K., Lapierre, L. A., and Goldenring, J. R. (2001) J. Biol. Chem. 276, 39067-390751). Here, we characterized the specificity of the interaction between Rab11-FIP2 and Rab11 and report that it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. We demonstrate that the COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself was insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, caused tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 was also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking.

The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Here, we characterise several of these proteins with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. We now demonstrate that two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4 and show that several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end which includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. We identify a new (sixth) member of the protein family, which we propose to name Rab11-FIP4, and report the family evolutionary complexity and chromosomal distribution. Furthermore, we propose that the ability of these proteins to bind each other will be important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity., ((c)2002 Elsevier Science (USA).)

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.

Rab4 belongs to the Rab family of small GTPases involved in the regulation of intracellular transport, and has been localized to early endosomes. We have employed the yeast two-hybrid system to identify proteins that specifically interact with Rab4AQ67L, a GTPase-deficient mutant form of Rab4A. Screening a mouse embryo cDNA library identified a clone (M449) that interacted with Rab4A in a nucleotide-dependent fashion. Data base searches identified this clone as the mouse cytoplasmic dynein light intermediate chain-1 (LIC-1). Based on this finding, the full-length equivalent human cytoplasmic dynein LIC-1 was isolated by PCR. When Rab4A was overexpressed together with either M449 or dynein LIC-1 in HeLa cells, the proteins were found to colocalize in the perinuclear region. We characterize the localization of both overexpressed human dynein LIC-1 and the endogenous protein with respect to microtubules and show that it concentrates to the microtubule-organizing center and mitotic spindle. Additionally, GFPRab4A endosomes localize to microtubules and are redistributed by nocodazole treatment. This is the first described interaction between cytoplasmic dynein, a retrograde motor protein, and a Rab protein., (Copyright 2001 Academic Press.)