Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators

Abstract

Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab escort protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated proteins is complicated by their low solubility in aqueous solutions. Here, we used expressed protein ligation to construct a semisynthetic fluorescent analogue of prenylated Rab7, Rab7-NBD-farnesyl. This molecule is soluble in the absence of detergent but is otherwise similar in its behavior to naturally prenylated Rab7 GTPase. To obtain information on the interaction of natively mono- and diprenylated Rab7 GTPases with REP and GDI molecules, we stabilized the former molecules in solution by using the beta-subunit of Rab geranylgeranyl transferase, which we demonstrate to function as an unspecific chaperone of prenylated proteins. Using competitive titrations of mixtures of natively prenylated and fluorescent Rab, we demonstrate that monogeranylgeranylated Rab7 binds to the REP protein with a K(d) value of approximately 70 pM. The affinity of doubly prenylated Rab7 is approximately 20-fold weaker. In contrast, GDI binds both prenylated forms of Rab7 with comparable affinities (K(d) = 1-5 nM) but has extremely low affinity to unprenylated Rab molecules. The obtained data allow us to formulate a thermodynamic model for the interaction of RabGTPases with their regulators and membranes and to explain the need for both REP and GDI in Rab function.

Scheme. 1.

Construction of prenylated fluorescent Rab7. (A) The fluorescent analogue of GG, NBD-farnesyl. (B) Synthesis of the fluorescent analogue of monogeranylgeranylated Rab, Rab7-NBD-farnesyl.

Fig. 1.

Gel filtration chromatography of semisynthetic Rab7-NF and analysis of its interaction with REP. (A) (Lower) Elution profile of Rab7-NF resolved on a Superdex 200 column and analysis of the resulting fractions by SDS/PAGE followed by Coomassie blue staining. (Upper) The dashed line represents absorbance at 280 nm, whereas the solid line represents fluorescence. The arrows in Upper indicate elution position of molecular mass standards in kilodaltons. Fractions 19–21 containing Rab7-NF at >80% purity were collected and used for further analysis (see SI Fig. 5A). (B) Emission spectra of Rab7-NF in the absence and in the presence of REP. (C) Titration of REP to a 10 nM solution of Rab7-NF. (D) Titration of GDI to a 10 nM solution of Rab7-NF. The fluorescence of NBD was excited at 479 nm, and the emission was collected at 525 nm. K_d values were obtained by fitting the data to the solution of a quadratic equation (see SI Appendix).

Fig. 2.

Solubilization of monogeranylgeranylated Rab7 by the β-subunit of RabGGTase and its interaction analysis with REP and GDI. (A) Gel filtration chromatography of Rab7-G:βGGT on a Superdex 200 column (Upper) and SDS/PAGE analysis of resulting fractions (Lower). (B) MALDI-TOF analysis of Rab7-G:βGGT complex (M_calc = 23,660 Da for Rab7-G). (C and D) Titration of REP (C) or GDI (D) to a mixture of 10 nM Rab7-G:βGGT and 10 nM Rab7-NF. The data were fitted by numerical simulation and fitting to a competitive binding model to give K_d values for the interaction of Rab7-G with REP or GDI. (E and F) Titration of REP (E) or GDI (F) to a mixture of 25 nM Rab7-NF and 25 nM Rab7d-GG:BSA (λ_ex/em:479/525 nm). The data were fitted to a competitive binding model to give K_d values of Rab7d-GG for REP and GDI, where K_d values of 0.22 nM and 21 nM of Rab7-NF for REP and GDI, respectively, were fixed.

Fig. 3.

Comparison of lipid binding sites of GDI and REP molecules in complex with prenylated Rab GTPases. (A) Monoprenylated Ypt1:GDI complex (1UKV). (B) Diprenylated Ypt1:GDI complex (2BCG). (C) Monoprenylated Rab7:REP complex (1VG0). (D) Unprenylated Rab7Δ22:REP complex (1VG9). The complexes were optimally superimposed, and the domains II were sliced to expose the lipid-binding site. All molecular manipulations including generation of images were performed with ICM Browser Pro (Molsoft, Redmond, WA).

Fig. 4.

Model for the extraction of Rab from membranes by GDI or REP. (A) Initial recognition of the membrane associated Rab. (B) Formation of the membrane-bound Rab:REP/GDI complex. (C) Translocation of the lipid moiety to the GDI/REP and release form the membrane.