Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling

Abstract

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.

Figure 1

Structure of the doubly prenylated Ypt1:GDI complex. (A) Ribbon representation of GDI (blue) bound to Ypt1 (yellow). Domain I (dark blue), domain II (light blue), the Rab-binding platform (red), the C-terminus-binding region (CBR gold) and the mobile effector loop (MEL green) of GDI are highlighted. The Switch I and II regions of Ypt1 are highlighted in green and grey, respectively. The helices composing domain II of GDI are marked by letters. The isoprenoid moieties 1 (red) and 2 (dark yellow) are displayed in ball-and-stick representation. The GDP (atomic colours) and Mg²⁺ (magenta) ion are shown in the nucleotide-binding pocket in ball-and-stick and space filling representations, respectively. Unless otherwise indicated, this and other figures were prepared using ICM (Molsoft LLC). (B) Plot of a sigma A-weighted F_o–F_c map contoured at 2σ (red) or at 0.6σ (black) in the region of the geranylgeranyl. The maps were generated after several refinement rounds omitting the GG groups. The picture was generated with BobScript and RASTER3D (Merritt and Murphy, 1994; Esnouf, 1997). (C) Domain II of GDI from Ypt1GG:GDI complex displayed in ribbon representation (grey); the secondary structure elements are denoted as in (A). The last visible residues of the Ypt1 C-terminus and of the MEL of GDI are coloured blue and green, respectively. The geranylgeranyl moieties 1 (red) and 2 (blue) filling the lipid binding site are displayed in ball-and-stick representation.

Figure 2

Organisation of the lipid binding site of the doubly prenylated Ypt1:GDI complex. (A) Surface representation of domain II of GDI. The MEL is coloured in green. The C-terminus of Ypt1 is displayed as a yellow worm. The geranylgeranyl moieties are displayed as in Figure 1C. (B) Surface representation of the doubly prenylated Ypt1:GDI complex. The MEL is coloured in green, while the geranylgeranyl moieties 1 and 2 are coloured red and blue, respectively. (C) Same as in (B) but solvent-exposed hydrophobic residues are coloured in yellow and geranylgeranyl 2 is displayed in ball-and-stick representation coloured in blue. (D) Superimposition of mono- and diprenylated Ypt1:GDI complexes. GDI from the diprenylated complex is displayed as in (A), but helix D forming the left wall of the lipid binding side is removed. Geranylgeranyl moieties of the doubly prenylated complex are displayed as in (A) and the geranylgeranyl of the monoprenylated Ypt1:GDI complex is displayed in ball-and-stick representation and coloured in green. (E) Superposition of the geranylgeranyl moieties of the mono- and diprenylated Ypt1:GDI complexes. The colouring is the same as in (D) but the carbon atoms are numbered.

Figure 3

Model for the GDI-mediated Rab/Ypt interaction with the putative Rab receptors and membranes. (A) GDI-mediated delivery of prenylated RabGTPases to the membrane is proposed to involve docking of the Rab:GDI complex with a putative membrane Rab recruitment/GDF via a protein:protein interaction (2). The docked complex undergoes a conformational change, which leads to the transfer of the first and then the second geranylgeranyl moiety into the membrane and subsequently to the release of the Rab C-terminus from the CBR (3 and 4). Finally, GDI is released into the cytosol and the Rab protein enters its functional cycle. (B) GDI-mediated extraction of prenylated RabGTPases from the membrane. Initial recognition is mediated by the low-affinity interaction of the Rab-binding platform with the CBR of GDI (7). This leads to the positioning of GDI domain II on the lipid bilayer over the buried geranylgeranyl moieties of the Rab protein (8). The geranylgeranyl lipids are transferred from the membrane to the lipid binding sites on GDI in two consecutive steps (9 and 10), leading to dissociation of the complex from membrane (11).

Figure 4

Interaction of GDI and Mrs6 with unprenylated and membrane-bound Ypt proteins. (A) ITC titration of Ypt1 with increasing concentrations of GDI. Fitting of the data led to a K_d value of 36 μM. (B) Same as in (A) but with Ypt7 (K_d=6 μM). (C) A representative fluorescence titration of dans_Ypt1 with Mrs6. The concentration of dans_YPT1 was 240 nM. The fluorescence of the dansyl group was excited at 280 nm via FRET from tryptophanes and the emission was collected at 490 nm. The data were corrected for unspecific fluorescence increase and analysed as described under ‘Materials and methods', leading to a K_d value of 37 nM. (D) Extraction of Ypt1 from S. cerevisiae membranes with yeast GDI. The isolated membrane fraction was incubated with various concentrations of recombinant GDI and the fraction of membrane-associated Ypt1 was determined by Western blotting as described in ‘Materials and methods'. (E) Same as in (D) but the membranes were incubated with recombinant Mrs6. Complete extraction of Ypt1 at high concentrations is not in contradiction to the model calculations, since these were made for specific values of the effective membrane concentration and the affinity of geranylgeranyl residues for membranes, which are not likely to apply here. (F) Unprenylated Ypt7 is able to interfere with the ability of GDI to extract membrane-bound Ypt1. S. cerevisiae membranes were incubated with 120 nM GDI and increasing concentrations of Ypt7. The sample in lane 6 was incubated with 30 μM of Ypt7 and no GDI, while the sample in lane 7 was incubated with buffer alone.

Figure 5

Quantitative simulation of the extraction of prenylated Rabs by GDI/REP based on a simple model. (A) Two-step binding of Rab to GDI/REP. The affinity between the two proteins would be given by K_GDI(1+K_dock). (B) Extension of the model to extraction from a membrane. This includes a step in which the dissociation of the C-terminal prenyl groups from the lipid bilayer occurs in a discrete step, which is mechanistically unrealistic but thermodynamically sound given coupling to the next step (docking of the lipid onto its GDI/REP binding site). (C) Simulation of the dependence of the degree of extraction of Rab as a function of the GDI/REP concentration using the constants given. For simulation, a value of 1 μM has been chosen for the effective membrane concentration, together with a value of 1 nM for the affinity of the prenylated C-terminus of Rab for the membrane. While these might not be an accurate reflection of the situation in reality, different values will have identical effects on both GDI and REP extraction efficiencies, but will not affect their relative efficiencies. (D) Same as in (C) but with a different set of constants differing less dramatically for GDI and REP.