Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia.

Ras related protein (Rab5a) is one of the most important member of the Rab family which regulates the early endosome fusion in endocytosis, and it also helps in the regulation of the budding process. Here, for the first time we report a simple and reproducible method for the purification of the Rab5a from a medicinal plant Tinospora cordifolia. We have used weak cation-exchange (CM-Sepharose-FF) followed by gel-filtration chromatography. A purified protein of 22-kDa was observed on SDS-PAGE which was identified as Rab5a using MALDI-TOF/MS. Our purification procedure is fast and simple with high yield. The purified protein was characterized using circular dichroism for the measurement of secondary structure followed by GdmCl- and urea-induced denaturation to calculate the values of Gibbs free energy change (ΔGD), ΔGD°, midpoint of the denaturation Cm, i.e. molar GdmCl [GdmCl] and molar urea [Urea] concentration at which ΔGD=0; and m, the slope (=∂ΔGD/∂[d]) values. Furthermore, thermodynamic properties of Rab5a were also measured by differential scanning calorimeter. Here, using isothermal calorimeteric measurements we further showed that Rab5a binds with the GTP. This is a first report on the purification and biophysical characterization of Rab5a protein from T. cordifolia.

Determination of Rab5 activity in the cell by effector pull-down assay.

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

Identification of the Rab5 binding site in p110ß: assays for PI3Kß binding to Rab5.

Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110ß, and p110δ), p110ß has unique properties. Unlike the other family members, p110ß directly binds to Gßγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110ß binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110ß signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110ß, and discusses methods for the analysis of p110ß-Rab5 interactions.

Rab5 affinity chromatography without nonhydrolyzable GTP analogues.

Rab5 is an important small GTPase involved in endocytosis and membrane trafficking. Rab5-binding proteins can be identified using Rab5 affinity chromatography with nonhydrolyzable GTP analogues such as GTPgammaS or GppNHp. However, this method requires significant quantities of the GTP analogue and is thus time-consuming and expensive. In the present report we show a faster and more cost-effective method that does not use a GTP analogue but uses constitutively the active Rab5 mutant (Rab5Q79L) as a ligand. To validate this method, the binding of EEA-1 was confirmed and several novel Rab5-binding proteins were also identified by 2-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).

An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway.

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

Purification and analysis of RIN family-novel Rab5 GEFs.

The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.

Purification and functional analyses of ALS2 and its homologue.

ALS2 is a causative gene product for a form of the familial motor neuron diseases. Computational genomic analysis identified ALS2CL, which is a novel protein highly homologous to the C-terminal region of ALS2. Both proteins contain the VPS9 domain, which is a hallmark for all known members of the guanine nucleotide exchange factors for Rab5 (Rab5GEF), and are known to act as novel factors modulating the Rab5-mediated endosome dynamics in the cells. It has also been reported that oligomerization of ALS2 is one of the fundamental features of its biochemical and physiological function involving endosome dynamics. This chapter describes methods, including purification of the recombinant ALS2 and ALS2CL, and Rab5GEF assay, which have been utilized to clarify the molecular function for ALS2 and ALS2CL.

Assay and stimulation of the Rab5 GTPase by the p85 alpha subunit of phosphatidylinositol 3-kinase.

Rab5 is a small monomeric GTPase involved in regulating vesicle fusion events during receptor-mediated endocytosis. During endocytosis of the activated platelet-derived growth factor receptor, phosphatidylinositol 3-kinase (PI3K) remains associated with the receptor. We have found that the p85 alpha subunit of PI3K binds directly to Rab5 and possesses GTPase-activating protein (GAP) activity toward Rab5. We describe two methods used to characterize the GAP activity of p85 toward the Rab5 protein. The first method is a steady-state GAP assay, used to show that the p85 alpha protein has GAP activity toward Rab5. The second method is a single turnover GAP assay and measures changes in the catalytic rate of Rab5 GTP hydrolysis with or without the p85 alpha protein.

Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.

Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.