A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.     

A role for the Rab6A' GTPase in the inactivation of the Mad2-spindle checkpoint

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A', regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A' function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150(Glued), a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A' likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A', through its interaction with p150(Glued) and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.     

Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport

Rab6 GTPase regulates intracellular transport at the level of the Golgi complex. Using the yeast two-hybrid screen, we have isolated two clones that specifically interact with the three isoforms of Rab6 present in mammalian cells (Rab6A, A' and B). The cDNAs encode two proteins of 976 and 1120 amino acids (calculated molecular mass of 112 and 128 kDa, respectively) that we named Rab6IP2A and Rab6IP2B (for Rab6 Interacting Protein 2). The two proteins likely correspond to spliced variants of the same gene. Rab6IP2s have no significant homology with other known proteins, including Rab effectors or partners. They are ubiquitously expressed, mostly cytosolic and found in high molecular mass complexes in brain cytosol. We show that Rab6IP2s can be recruited on Golgi membranes in a Rab6:GTP-dependent manner. The overexpression of any form of Rab6IP2 has no detectable effect on the secretory pathway. In contrast, the retrograde transport of the Shiga toxin B subunit between the plasma membrane and the Golgi complex is partly inhibited in cells overexpressing the Rab6-binding domain of Rab6IP2. Our data suggest that Rab6IP2s is involved in the pathway regulated by Rab6A'.     

Alternative splicing of the human Rab6A gene generates two close but functionally different isoforms

Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.     

Interaction of the DYNLT (TCTEX1/RP3) light chains and the intermediate chains reveals novel intersubunit regulation during assembly of the dynein complex

The cytoplasmic dynein 1 cargo binding domain is formed by five subunits including the intermediate chain and the DYNLT, DYNLL, and DYNLRB light chain families. Six isoforms of the intermediate chain and two isoforms of each of the light chain families have been identified in mammals. There is evidence that different subunit isoforms are involved in regulating dynein function, in particular linking dynein to different cargoes. However, it is unclear how the subunit isoforms are assembled or if there is any specificity to their interactions. Co-immunoprecipitation using DYNLT-specific antibodies reveals that dynein complexes with DYNLT light chains also contain the DYNLL and DYNLRB light chains. The DYNLT light chains, but not DYNLL light chains, associate exclusively with the dynein complex. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that both members of the DYNLT family are capable of forming homodimers and heterodimers. In addition, both homodimers of the DYNLT family bind all six intermediate chain isoforms. However, DYNLT heterodimers do not bind to the intermediate chain. Thus, whereas all combinations of DYNLT light chain dimers can be made, not all of the possible combinations of the isoforms are utilized during the assembly of the dynein complex.     

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking

The closely related Rab6 isoforms, Rab6A and Rab6A', have been shown to regulate vesicular trafficking within the Golgi and post-Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A' biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A' hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B-subunit along the endocytic pathway, and causes defects in Golgi- associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A' is required for cell cycle progression through mitosis and identify Ile(62) as a key residue for uncoupling Rab6A' functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A' perform different functions within the cell and suggests a novel role for Rab6A' as the major Rab6 isoform regulating previously described Rab6-dependent transport pathways.     

Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'

Ricin is transported from early endosomes and/or the recycling compartment to the trans-Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo- and vector-based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A' mRNA was simultaneously up-regulated, the inhibition of ricin sulfation was abolished, indicating that the up-regulation of Rab6A' may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A' gave approximately 40% reduction in ricin sulfation. The up-regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A' function. The depletion of both Rab6A and Rab6A' gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A' seem to be involved in the transport of ricin from endosomes to the Golgi apparatus.     

Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.     

Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase

Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.     

Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome

The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.     

Rab11-FIP3 binds dynein light intermediate chain 2 and its overexpression fragments the Golgi complex

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.     

Rab6-interacting protein 1 links Rab6 and Rab11 function

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.     

Giantin interacts with both the small GTPase Rab6 and Rab1

The interaction of small GTPases of the Rab family and coiled coil proteins of the golgin family has been reported for example for the Rab1 GTPase and p115, GM130 and Giantin. We now show that Rab6A, a GTPase that controls retrograde trafficking within the Golgi back to the endoplasmic reticulum is also able to bind to Giantin in vivo and in vitro pointing to an interesting complex formation between Giantin and two different Rab GTPases. In Saccharomyces cerevisiae a genetic interaction between Ypt1 and Ypt6 has already been demonstrated, but in this paper we were able to describe that the mammalian Rab GTPases are able to interact on the same golgin protein, Giantin.     

Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.     

Identification of a novel region of the cytoplasmic Dynein intermediate chain important for dimerization in the absence of the light chains

Cytoplasmic dynein is the multisubunit protein complex responsible for many microtubule-based intracellular movements. Its cargo binding domain consists of dimers of five subunits: the intermediate chains, the light intermediate chains, and the Tctex1, Roadblock, and LC8 light chains. The intermediate chains have a key role in the dynein complex. They bind the three light chains and the heavy chains, which contain the motor domains, but little is known about how the two intermediate chains interact. There are six intermediate chain isoforms, and it has been hypothesized that different isoforms may regulate specific dynein functions. However, there are little data on the potential combinations of the intermediate chain isoforms in the dynein complexes. We used co-immunoprecipitation analyses to demonstrate that all combinations of homo- and heterodimers of the six intermediate chains are possible. Therefore the formation of dynein complexes with different combinations of isoforms is not limited by interaction between the various intermediate chains. We further sought to identify the domain necessary for the dimerization of the intermediate chains. Analysis of a series of truncation and deletion mutants showed that a 61-amino-acid region is necessary for dimerization of the intermediate chain. This region does not include the N-terminal coiled-coil, the C-terminal WD repeat domain, or the three different binding sites for the Tctex1, LC8, and Roadblock light chains. Analytical gel filtration and covalent cross-linking of purified recombinant polypeptides further demonstrated that the intermediate chains can dimerize in vitro in the absence of the light chains.     

COPI recruitment is modulated by a Rab1b-dependent mechanism

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of beta-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.     

Rab6 interacts with the mint3 adaptor protein

The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.     

Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex

The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein-dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein-dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)-Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein-dynactin complex controls COPI-independent Golgi-ER transport.