Biochemical Society Transactions

Cellular Cytoskeletal Motor Protein

Rab GTPases and microtubule motors

Conor P. Horgan, Mary W. McCaffrey

Abstract

Rab proteins are a family of small GTPases which, since their initial identification in the late 1980s, have emerged as master regulators of all stages of intracellular trafficking processes in eukaryotic cells. Rabs cycle between distinct conformations that are dependent on their guanine-nucleotide-bound status. When active (GTP-bound), Rabs are distributed to the cytosolic face of specific membranous compartments where they recruit downstream effector proteins. Rab–effector complexes then execute precise intracellular trafficking steps, which, in many cases, include vesicle motility. Microtubule-based kinesin and cytoplasmic dynein motor complexes are prominent among the classes of known Rab effector proteins. Additionally, many Rabs associate with microtubule-based motors via effectors that act as adaptor molecules that can simultaneously associate with the GTP-bound Rab and specific motor complexes. Thus, through association with motor complexes, Rab proteins can allow for membrane association and directional movement of various vesicular cargos along the microtubule cytoskeleton. In this mini-review, we highlight the expanding repertoire of Rab/microtubule motor protein interactions, and, in doing so, present an outline of the multiplicity of transport processes which result from such interactions.

  • dynactin
  • dynein
  • kinesin
  • microtubule motor
  • Rab GTPase
  • Rab11

Background

Compartmentalization within eukaryotic cells necessitates highly regulated transport of cellular material within and between intracellular organelles. This transport usually involves the formation of transport intermediates in the form of membrane-bound vesicles or tubules that bud from donor compartments and are transported along the microtubule and/or actin cytoskeleton until they reach an acceptor compartment with which they dock and fuse. This mechanism of intracellular transport is conserved from yeast to humans and involves multiple conserved protein families such as coat proteins, motor proteins, tethering factors and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) [1]. Although these proteins are required for distinct and sequential trafficking steps (coats for budding, motors for motility, tethers for docking and SNAREs for fusion), members of the Rab GTPase family have emerged as overriding regulators of all stages of intracellular trafficking [2].

Rab GTPases

Rabs are a family of monomeric G proteins (guanine-nucleotide-binding proteins) comprising more than 60 members, which constitute the largest branch of the Ras GTPase superfamily [2]. In mammals, many Rabs are ubiquitously expressed while others display tissue-specific patterns of expression [2]. Rab proteins are post-translationally modified by isoprenylation, whereby thioester bonds allow the covalent linkage of one or two GGPP (geranylgeranyl pyrophosphate) groups to short cysteine motifs (-CC, -CXC) at the C-terminus. These hydrophobic moieties facilitate reversible membrane association of the Rab [2]. In their active states (GTP-bound; discussed below), Rabs are distributed to the cytosolic face of intricately specific, although sometimes overlapping, membranous compartments in both the endocytic and biosynthetic pathways, where they regulate each of the stages of intracellular trafficking, including vesicle formation, motility, docking and fusion.

Like all members of the Ras superfamily, Rab proteins cycle between active and inactive conformations, which are dependent on their guanine-nucleotide-bound status. This conformational cycle involves changes in two key variable regions in the Rabs termed switch I and switch II [2]. When GDP-bound, Rab proteins are inactive and are usually distributed to the cytosol where they complex with chaperone proteins termed GDIs (guanine-nucleotide-dissociation inhibitors). GDIs interact with the isoprenylated C-terminus of the Rabs and block their dissociation from GDP. Although it is not entirely clear as to how Rab proteins achieve their very heterogeneous and specific membrane targeting, it appears that membrane-bound GDFs (GDI-displacement factors) play a role in recognizing specific Rab-GDP–GDI complexes, promoting GDI release and thus facilitating insertion of the Rab-attached isoprenoid groups into the membrane [2]. This highly specific targeting event is likely to involve the C-terminal hypervariable region of the Rab [2]. Once on an appropriate membrane, GEFs (guanine-nucleotide-exchange factors) facilitate exchange of GDP for GTP. When GTP-bound, the Rab is active and recruits downstream ‘effector’ proteins through which its function is elicited. GAPs (GTPase-activating proteins) then stimulate the intrinsic GTPase activity of the Rab that hydrolyses the terminal phosphate of the GTP, thus inactivating the Rab, with consequential disengagement from its downstream effectors. Finally, the Rab is extracted from the membrane by GDI, returning the GDP-bound inactive Rab to the cytosol [2].

As mentioned, the ability of GTP-bound Rabs to co-ordinate their multitude of cellular functions primarily arises from their ability to recruit downstream effector proteins. Identified Rab effectors are a heterogeneous collection of molecules that include coat proteins, sorting adaptors, tethering factors, kinases, phosphatases, SNAREs, scaffold/linker proteins and actin and microtubule-based molecular motor proteins.

Microtubules and microtubule motor proteins

In all eukaryotic cells, there exists a filamentous network of cellular proteins, termed the cytoskeleton, which primarily comprises three distinct classes of fibres: microfilaments, microtubules and intermediate filaments. The role of the cytoskeleton is multifaceted, but some of its key functions include giving the cell rigidity and strength that helps maintain cell shape, and to provide intracellular tracks that allow for the directed movement of organelles and their transport intermediates during intracellular trafficking processes.

Microtubules, one of the major components of the cytoskeletal network, are hollow cylindrical fibres of approximately 25 nm in diameter that are formed by the polymerization of α,β-tubulin heterodimers [3]. The tubulin heterodimers polymerize head-to-tail into protofilaments that bundle in a parallel fashion and give rise to polar microtubules with fast-growing plus-ends (β-subunit exposed) and slow-growing minus-ends (α-subunit exposed) [3]. This polarity is crucially significant in determining the directional movement of motor proteins along the microtubule surface. Microtubules are nucleated and organized from MTOCs (microtubule-organizing centres), such as the centrosome and basal bodies, and continually undergo assembly and disassembly within the cell, making the microtubule network temporally and spatially dynamic [3]. This dynamic nature of microtubules facilitates the changing needs of cells as they perform disparate cellular tasks such as long-range transport of vesicles or cellular organelles during interphase or segregating chromosomes during cell division.

Two protein superfamilies have been identified that utilize microtubules as tracks along which to move intracellular material in eukaryotic cells: the kinesin superfamily proteins (alternatively known as KIFs) and dyneins. In humans, the gene products of 45 separate KIF genes constitute the kinesin superfamily and these have been subdivided into 15 multi-component kinesin families termed kinesin-1–kinesin-14B [4]. Kinesin structure varies, but the initial kinesin to be discovered, conventional kinesin, is a tetramer consisting of two KHCs (kinesin heavy chains) that contain the globular motor domains, and two KLCs (kinesin light chains) that connect the complex to its cargo [5]. Being ATPases, kinesins utilize ATP hydrolysis to drive conformational changes in the protein that generates motile force. The majority of identified kinesins act as plus-end-directed motors although some move material towards the microtubule minus-end [4].

On the other hand, the major minus-end-directed microtubule motors in eukaryotic cells are multi-component ATPases termed dyneins [6]. Dyneins can be divided into two groups: axonemal dynein, which is required for the motility of cilia and flagella, and cytoplasmic dyneins, which are essential for many cellular processes. A total of 14 distinct cytoplasmic dynein subunits have been identified in humans, and, depending on which cytoplasmic dynein HCs (heavy chains) the cytoplasmic dynein complex is formed around, cytoplasmic dyneins can be further divided into cytoplasmic dynein-1 and cytoplasmic dynein-2 (for a synopsis of the standardized dynein nomenclature, see [7]). Cytoplasmic dynein-1, the primary cytoplasmic dynein in eukaryotes, is critical for multiple cellular processes such as intracellular trafficking and mitosis, while cytoplasmic dynein-2 has roles in generation and maintenance of cilia and organization of the Golgi complex [812]. Cytoplasmic dynein-1 complexes are formed around a cytoplasmic dynein-1 HC homodimer and also comprise IC (intermediate chain), LIC (light intermediate chain) and LC (light chain) subunits [9,13]. In addition, cytoplasmic dynein-1 usually requires the activity of an accessory protein complex termed dynactin (otherwise known as dynein activator complex). Dynactin serves as an adaptor for interaction of cytoplasmic dynein-1 with many of its cargo organelles, and enhances the processivity of cytoplasmic dynein and, interestingly, kinesin-2 [1416].

Regulation of microtubule motors by Rab GTPases

During the 1990s, the initial publications documenting the involvement of Rabs in the regulation of microtubule motors emerged, and since then, numerous reports have been forthcoming. Indeed, it is now clear that Rab GTPases perform central roles in motor protein regulation and are key determinants of cells ability to attach their membranous cargo to microtubule motor proteins and thus the microtubule cytoskeleton.

Rab–kinesin interactions

Among the first descriptions of Rab-mediated membrane-association of kinesin motors was a study by Echard et al. [17], which described a direct and GTP-dependent interaction between Rab6A and KIF20A (kinesin-6 subunit). Later, this interaction was shown to be required for successful cleavage furrow formation and cytokinesis during cell division [18]. A further account of a direct Rab–kinesin interaction was recently reported by Ueno et al. [19], whereby Rab14 was found to directly associate with KIF16B (kinesin-3 subunit) and shown to be required for Golgi-to-endosome trafficking of the FGFR (fibroblast growth factor receptor) during embryonic development.

While the aforementioned studies reveal details of direct interactions between Rabs and kinesins, published data from the literature indicate that Rab proteins more frequently associate with their cognate kinesin motors via adaptor/linker proteins that simultaneously bind the Rab and the kinesin motor. Indeed, Rabs 3, 6, 9, 11 and 27, and in many cases their closely related Rab-subfamily members, have all been shown to control various intracellular trafficking events through binding to different kinesin complexes via distinct downstream adaptor proteins (see Table 1). For example, Rab3A associates with KIF1Bβ/KIF1A (kinesin-3 subunits) via DENN (differentially expressed in normal and neoplastic cells)/MADD [MAPK (mitogen-activated protein kinase)-activating death domain] and is required for axonal transport of Rab3A-containing vesicles in neuronal cells [20]; and Rab11A regulates endosomal trafficking events by associating with KIF3B (kinesin-2 subunit) via Rip11 (Rab11-interacting protein) [alternatively known as FIP (Rab11 family-interacting protein) 5] [21]. Interestingly, Rab27A and Rab27B associate with KIF5/KLC1 (kinesin-1 subunit) via two linker proteins, Slp1 (synaptotagmin-like protein-1) and CRMP-2 (collapsin response mediator protein-2), to regulate axonal transport of neurotrophin receptor-containing vesicles [22].

View this table:
Table 1 Rab GTPase interactions with KIFs

SKIP, SifA and kinesin-interacting protein; Slp1, synaptotagmin-like protein-1.

Some Rabs have been shown to associate with multiple kinesin complexes to regulate distinct trafficking processes. For example, Rab6A, which as indicated above, directly binds KIF20A (kinesin-6 subunit), also associates with kinesin-1 and kinesin-3 via BICD (Bicaudal D homologue-2) and BICDR-1 (BICD-related protein 1) respectively; and these interactions have been demonstrated to be required for separate trafficking processes in distinct cellular contexts [23,24].

Rab–dynein interactions

The initial studies illustrating that Rab GTPases direct a range of dynein-mediated intracellular trafficking steps began to emerge in 2001. Our group reported that Rab4A, a sorting endosome Rab, directly interacts with dynein LIC1 [DYNC1LI (dynein, cytoplasmic 1, light intermediate chain) 1] in a GTP-dependent manner [25]. Later that year, Jordens et al. [26] reported that Rab7 associates with cytoplasmic dynein-1 through the binding of RILP (Rab-interacting lysosomal protein), a Rab7 effector protein, to the dynactin p150/glued subunit to control late endosomal transport.

More recently, our group found that Rab11A controls trafficking processes from sorting endosomes to the endosomalrecycling compartment (also called recycling endosome) by linking to the cytoplasmic dynein-1 LIC1 and LIC2 subunits (DYNC1LI1 and DYNC1LI2) via the Rab11 effector protein FIP3 [27,28]. Interestingly, as LIC1 and LIC2 are believed to associate with cytoplasmic dynein-1 HCs in a mutually exclusive manner, the implication of these findings is that Rab11A and FIP3 can associate with at least two distinct cytoplasmic dynein-1 motor complexes [29,30]. These Rab11–cytoplasmic dynein-1 associations appear to be conserved in fruitflies as Nuf (nuclear fallout), the FIP3 orthologue in Drosophila melanogaster, links Rab11 to cytoplasmic dynein-1 via dynein LIC (the DYNC1LI1 and DYNC1LI2 orthologue in D. melanogaster) during endosomal shuttling in mechanosensory bristles [31].

Nonetheless, members of the Rab6 subfamily are the Rabs with the most known distinct molecular links with cytoplasmic dynein-1 complexes. Rab6A, Rab6A′ and Rab6B all directly bind the cytoplasmic dynein-1 Roadblock LC-1 subunit [DYNLRB1 (dynein, light chain, roadblock-type 1)]; and Rab6A, and in one case Rab6B, can associate with cytoplasmic dynein-1 via binding the dynactin p50/dynamitin and p150/glued subunits (see Table 2) [3236]. As is the case with the Rab6–kinesin interactions outlined above, these functional associations between Rab6-subfamily members and cytoplasmic dynein-1 complexes facilitate distinct trafficking steps along the biosynthetic pathway.

View this table:
Table 2 Rab GTPase interactions with cytoplasmic dynein-1 or dynactin subunits

Conclusion

Over the last decade, much information regarding functional cellular interplay between Rab GTPases and microtubule motor complexes has been forthcoming. Nevertheless, it seems very likely that many further interactions remain to be identified. This supposition is predicated on the fact that, for the majority of Rabs, no functional associations with motor proteins have been described. Demarcation of such Rab–motor associations and elucidation of the specific cargoes transported as a consequence of such interactions is an area of key interest in our laboratory and represents exciting challenges for the future.

Funding

This work was supported by a Science Foundation Ireland Investigator Grant [grant number 05/IN.3/B859] and a Science Foundation Ireland Research Frontiers Grant [grant number 08-RFPNSC1499].

Acknowledgments

We thank Sara Hanscom and Eoin Kelly for helpful suggestions and critically reading this paper before submission.

Footnotes

  • Cellular Cytoskeletal Motor Proteins: A Biochemical Society/Wellcome Trust Focused Meeting held at Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K., 30 March–1 April 2011. Organized and Edited by Folma Buss (Cambridge, U.K.) and John Kendrick-Jones (MRC Laboratory of Molecular Biology, Cambridge, U.K.).

Abbreviations: BICD, Bicaudal D homologue; BICDR-1, BICD-related protein-1; CRMP-2, collapsin response mediator protein-2; DENN, differentially expressed in normal and neoplastic cells; DYNC1LI dynein, cytoplasmic 1, light intermediate chain; DYNLRB1 dynein, light chain, roadblock-type 1; FIP, Rab11 family-interacting protein; GDI, guanine-nucleotide-dissociation inhibitor; HC, heavy chain; KIF, kinesin superfamily protein; KLC, kinesin light chain; LC, light chain; LIC, light intermediate chain; MADD, MAPK (mitogen-activated protein kinase)-activating death domain; Nuf, nuclear fallout; RILP, Rab-interacting lysosomal protein; Rip11, Rab11-interacting protein; Slp1, synaptotagmin-like protein-1; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor

References

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