Abstract
The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTP-bound Rab11a and Rab11b in vitro (50-100 nm affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nm) is not sufficient for FIP and GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.
Highlights
Rab proteins constitute the largest family of small monomeric GTPases and play an important role in membrane trafficking pathways
Rab11 Effector Proteins (FIPs) Form Homodimers—It has been previously suggested that FIPs can form homodimers and heterodimers, yet it remains unclear whether dimerization is dependent on Rab11 binding [26]
The binding was specific to Rip11 (Fig. 1A), as there was no binding observed with GST alone or with other FIP family member proteins, FIP2 (Fig. 1B) or FIP3 (Fig. 1A)
Summary
Plasmids and Reagents—Glutathione S-transferase (GST) gene fusion constructs were prepared by cloning GST-Rip, GST-FIP2, and GST-FIP3 into the baculovirus expression plasmid pAcGHLT-B (BD Biosciences) and Rab11a into pGEX-KG (Amersham Biosciences). Rab (8 M) was loaded in the sample cell (in phosphatebuffered saline containing 5 mM MgCl2 and 0.5 mM Gpp(NH)p or GDP; 1.426-ml volume) and titrated with Rip11-F1, FIP2-F1, GST-RCP-F1, and Rip11-F1 proteins in the same buffer The cells were permeabilized with phosphate-buffered saline containing 0.4% saponin, 2% fetal bovine serum, and 1% bovine serum albumin for 30 min, followed by incubation with rabbit anti-Rip or anti-Rab polyclonal antibody (Zymed Laboratories Inc.). Cells were incubated at 4 °C for 30 min, followed by internalization for 20 min at 37 °C in the continuous presence of Alexa 647-conjugated Tf. Cells were washed and incubated in complete medium supplemented with 50 g/ml unlabeled Tf for various times prior to fixation. The experiment was terminated as described above and analyzed on a BD Calibur flow cytometer (BD Biosciences) equipped with 488- and 647-nm lasers, gating for transfected cells (10,000 GFP-positive cells), and the amount of Tf internalized was determined
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