Function of Rabs
Rab proteins are a large family within the Ras superfamily. Rabs are GTPase proteins that cycle between a GTP-bound state in which they are able to bind effector proteins and a GDP-bound state in which they are inactive. Rabs facilitate protein trafficking from one compartment to another. There are numerous Rab proteins involved in various stages of protein trafficking.
Rab proteins regulate the tethering and/or docking of vesicles to target membranes including specific endocytic compartments and the plasma membrane. Fusion of secretory vesicles is regulated by Rab 3a and 27. Rab 8 localizes to the trans golgi network (TGN) and regulates the trafficking of proteins from the TGN to the plasma membrane. Rab 11 regulates neurotransmitter release in neurons as well as neurosecretion in PC 12 cells. Rab 11 predominantly localizes to the pericentriolar recycling endosomes and is thought to regulate trafficking of proteins from the recycling endosomes to the plasma membrane. One of the best studied transporters that undergo rapid plasma membrane fusion similar to DAT is the insulin-sensitive glucose transporter 4 (GLUT4). GLUT4 has striking similarities to DAT in its trafficking regulation and intracellular localization.
Structure of Rab
Crystallographic analyses have revealed that Rab proteins hold hypervariable N and C terminal regions, which may participate directly in protein-protein contacts with some effectors or regulatory proteins. The hypervariable carboxyl terminals consist of 35-40 amino acids, which have been implicated in subcellular targeting. Six different carboxyl-terminal motifs are known to date: XXXCC, XCCXX, XX- CXC, CCXXX, XXCCX and XCXXX, where X represents any amino acid. Rab proteins usually consist of two carboxyl-terminal cysteines involved in prenylation. Most Rab proteins are doubly geranylgeranylated (dual prenylation) at or near their C-termini which promotes their association with membranes. In particular, the geranylgeranyl groups are attached to one or more carboxy-terminal Cys residues, crucial in regulating membrane traffic.
Rab GTPases contain a guanine nucleotide fold (GTP active fold) consisting of a six stranded β-sheet, comprising five parallel strands and one antiparallel strand encompassed by five α helices. The elements necessary for guanine nucleotide and Mg2+ binding as well as GTP hydrolysis are found within the five loops that connect the α helices and the β strands. Regions involved in guanine-nucleotide binding and hydrolysis are the most conserved in all small GTPases.
Rab proteins additionally contain two important regions that allow them to adopt two different conformations, denoted switch I and switch II regions. Upon formation of ‘active Rab’ (GTP bound Rab), a broad hydrophobic interface forms between switch region I and II, resulting in ordered structural features that enable binding and response to effectors and regulators such as GDP/GTP exchange factors and GTPase activating proteins.
Recent sequence analysis has also revealed that Rab GTPases consist of five distinct amino acid stretches termed RabF regions (Rab family regions), that group within and around both switch regions. The RabF1 region is found within loop2/β of switch region I, while Rab -F2, -F3, -F4 and -F5 dwell within and around switch region II, between β3 and β4 sheets. Likewise, four highly conserved Rab subfamily regions termed RabSF have also been identified. RabSF1, RabSF3, and RabSF4 have all previously been known as the Rab complementary-determining regions I, II, and III and are required for specific binding of effector molecules
Functional role of Rab GTPases
Rab GTPases are known as key regulators of most vesicle trafficking events including vesicle formation, motility, tethering and fusion, as well as signaling. The importance of Rabs in regulating the diverse vesicle trafficking processes is represented by the high count of ‘different’ Rab proteins. Consistent with this, Rab proteins are localized to distinct intracellular compartments enabling specific membrane trafficking between these organelles.
During Rab mediated intracellular transport, it is important that each transport step involve Rab proteins bound to effectors (soluble proteins) to help drive their downstream functions. The effectors serve to regulate Rab activation, post-translational modification and intracellular localization.
Rabs regulate the formation of vesicles at the donor membrane, the movement of vesicles, tethering and docking of vesicles and their fusion with the acceptor membrane. Rab-mediated vesicle delivery is mediated in part by actin filaments and microtubules which regulate local and distant vesicle transport respectively. Microtubule-dependent traffic requires plus-end-directed motors of the kinesin superfamily or minus-end-directed motors like cytoplasmic dynein, which are regulated by Rab proteins. For instance, kinesin acts as a direct effector of Rab6 in vesicle transport, and is also indirectly regulated by Rab proteins. Proper tethering of vesicles to their target compartment and subsequent membrane fusion relies on the combined action of Rab proteins and their effectors. Previously, there have been examples where Rab5 and its effector EEA1 mediate clathrin-coated vesicle transport from the plasma membrane to the early endosomes and tethering/docking with early endosomes
Previous studies also implicate Rab GTPases as regulators of receptor recycling (i.e. transmembrane receptors including EGFR, integrins, and others). The Rab4 GTPase is responsible for the ‘short-loop’ recycling pathway while the Rab11 GTPase takes care of the ‘long-loop’ recycling pathway. In the short-loop recycling pathway, receptors such as integrin and transferrin return to the plasma membrane from the early endosomes. On the contrary, the long-loop recycling pathway involves the transport of receptors from the early endosomes to the perinuclear recycling compartment. Recycling of receptors as well as all membrane transport occurs via Rab and Rab-associated proteins.