UNC119 is required for G protein trafficking in sensory neurons

Abstract

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

Figure 1. Crystal structure of human UNC119

(A) Ribbon representation of the structure of human UNC119 (residues 57–237). Nine β strands (β1–β9), shown in red, create two β-sheets that splay apart at one end to create an opening to the cavity at the center of the β-sandwich. The N- and C-termini are marked N and C, respectively. The structure of the loop 108–123 connecting strands β2 and β3 could not be resolved. (B) Structure of UNC119 viewed after a 90° rotation around the vertical axis.

Figure 2. Interaction of UNC119 with Tα polypeptides

(A) Pulldown of rod and cone Tα with GST-UNC119 (representative Coomassie stained gel of four independent experiments). Bound polypeptides from bovine retina lysates were analyzed by SDS-PAGE. Lane 1, recombinant GST (G); Lane 2, pulldown with GST as a control (GC); Lane 3, GST-UNC119 pulldown (GU); Lane 4, GST-UNC119 fusion protein. An asterisk identifies the proteins pulled down by GST-UNC119. (B) Identification of peptides by LC-MS/MS. The 40 kDa polypeptides pulled down with GST-UNC119 were sequenced by LC-MS/MS. Identified peptide sequences, shown in red, were matched with rod and cone Tα. (C) GST-UNC119 pulldown of Tα from wild-type (lanes 1–4) and Gnat1^−/− retina (lanes 5–8). Lanes 1,2,5,6, input; lanes 3,7, control pulldowns with GST; lanes 4,8, pulldowns with GST-UNC119. Acylated Tα is pulled down (lane 4), but not farnesylated Tγ (lane 8). (D) GST-UNC119 pulldown of Tα and Tα(G2A) expressed in HEK cells. Lanes 1–3, input; lanes 4–6, pulldowns. Lanes 1,4, HEK cells expressing bovine Tα; Lanes 2,5, HEK cells expressing bovine Tα(G2A); Lanes 3 and 6, mouse retina lysates. Blot was probed with anti-Tα antibody. Note that UNC119 does not interact with non-acylated Tα(G2A). (E) Specificity of retina lysate pulldowns. Lanes 1–3, mouse retina lysates (input); lanes 4–6, retina lysate pulldowns; lane 4, GST control; lane 5, 10 µg GST-UNC119 was added; lane 6, same as lane 5 but with 0.1% Triton X-100 and 0.1% NP-40 (DT) present in the binding buffer. Top panel, blot probed with anti-Tα. Bottom panel, same blot probed with anti-GCAP1 antibody. Note that myristoylated GCAP1 does not interact with GST-UNC119.

Figure 3. UNC119 is an acyl-binding protein

(A) GST-UNC119 pulldowns and inhibition by an acylated N-terminal Tα peptide. Lane 1, retina lysate. Lanes 2–4, glutathione bead pellets of retina lysates that were incubated with GST-UNC119, in the absence of peptide (lane 2), the presence of lauroyl-GAGASAEEKH (lane 3), and in the presence of non-acylated GAGASAEEKH peptide (lane 4). Lanes 5–7, supernatants of 2–4. Note that lauroyl-GAGASAEEKH competes for binding (lane 3), but the non-acylated peptide did not (lane 4). (B) Isothermal titration calorimetry. Human UNC119 was titrated with G protein α-subunit N-terminal peptides. Red symbols, titration with N-terminal Tα peptide (lauroyl-GAGASAEEKH); black circles, titration with non-lauroylated GAGASAEEKH; green triangles, titration with myristoylated GAGASAEEKH; blue squares, titration with ODR-3 N-terminal peptide lauroyl-GSCQSNENSE. Lauroyl-GAGASAEEKH (red) and myristoyl-GAGASAEEKH (green) peptides were fit to a one-site model and bind with K_Ds of 0.54 µM ± 0.28 µM and 0.22 µM ± 0.14 µM, respectively. Lauroyl-ODR-3 (blue) binds more than one order of magnitude weaker (16.4 µM ± 3.0 µM). (C) Alignment of N-terminal peptides of mouse G protein α subunits, C. elegans G protein α-subunits GPA-13 and ODR-3, and Ca²⁺-binding proteins GCAP1, GCAP2 and recoverin. Peptides lacking Gly at position 2 cannot be myristoylated, therefore interaction with UNC119 through an acyl chain does not extend to all subfamilies of Gα.

Figure 4. The lipid binding pocket of UNC119

(A,B) Two orientations of UNC119 co-crystallized with the acylated Tα peptide in the UNC119 hydrophobic cavity. The lauroyl chain is shown in green, and the ten amino acids of the peptide are modeled in dark gray. In B, UNC119 is viewed after a 90° rotation around the vertical axis and the individual β-strands are labeled β1-9 in yellow. (C) Stereoview of UNC119 residues and key water molecules interacting with the lauroyl-GAGASAEEKH ligand. The hydrogen-bonding network (black dashed lines) limits the depth to which the Tα peptide can penetrate UNC119. Hydrogen bonds were included if the average of the bond length for all six molecules in the asymmetric unit was 3.2 Å or less and satisfied appropriate hydrogen bonding stereochemistry. UNC119 residues are shown in yellow, the lauroyl chain is green and the attached residues are colored dark gray. Figures were created with PyMOL (www.pymol.org).

Figure 5. UNC119 Interacts with Tα-GTP and Inhibits GTPase Activity

(A) Extraction of Tα from membranes by UNC119. Live mice were exposed to 10,000 lux/20 minutes driving transducin to the inner segments. Retina lysates in 1X PBS were incubated with either GST, mUNC119, GST and GTP, or mUNC119 and GTP, respectively. The soluble proteins (S) were separated from membrane-bound proteins (P) by centrifugation. Tα was detected by western blot using anti-Tα antibody. Tα elutes only in the presence of UNC119 and GTP. (B) Pulldown assays with light-adapted and dark-adapted mouse retinas. PBS/GTP supernatants from retinas of a light-adapted mouse (2,000 lux) and hypotonic supernatants from retinas of a dark-adapted mouse were used for pulldown assays, respectively. The proteins pulled down by GST or GST-UNC119 (pellet) and unbound proteins (supernatant) were analyzed by western blot using anti-Tα antibody. GST-UNC119 binds Tα^GTP (left), but not Tα^GDPTβγ (right). (C) GTPase activity of purified Tαβγ in the presence of ROS membranes. The activity of the reconstituted system (red line) corresponds to a rate of 1.5 mole GTP/min. Addition of BSA (light blue) has little effect, whereas addition of UNC119 (green) reduces the activity nearly to baseline. Baseline activity is caused by a low amount of Tαβγ still attached to the membranes (see inset). Inset, SDS-PAGE of purified native transducin (only Tα and Tβ subunits are shown), recombinant human UNC119 and depleted ROS membranes containing rhodopsin and a trace of transducin (only Tα is visible, Tβ co-migrates with rhodopsin).

Figure 6. Slow Return of Transducin to the Outer Segment after Intense Light Exposure

(A,B) Localization of Tα (green) in dark-adapted wild-type and Unc119^−/− retina. Mice were dark-adapted for at least 12 hours. Frozen sections were probed with anti-Tα antibody and FITC-linked secondary antibody. Note the presence of Tα in dark-adapted inner segments. (C–J) Mice were first exposed to intense light for 60 minutes, then dark-adapted for 0–24 hours. Frozen sections were probed with anti-Tα and FITC-linked secondary antibody. Note that Tα slowly returns to the wild-type outer segment, but is blocked in part from returning to the Unc119^−/− outer segment. OS, outer segment; IS, inner segment; ONL, outer nuclear layer. (K) Quantification of inner segment fluorescence at 0, 3, 6, and 24 hours after start of dark-adaptation. Fluorescence signal was quantified using ImageJ software. Each bar included three independent measurements. Error bars denote means ± SD.

Figure 7. Mislocalization of the G proteins ODR-3 and GPA-13 in a C. elegans unc-119(ed3) mutant

(A–D) Wild-type(A,C) and unc-119 mutant (B,D) C. elegans were stained with an anti-ODR-3 (A,B) and anti GPA-13 (C,D) antibody. Mislocalization of ODR-3 and GPA-13 to the olfactory cell bodies and axons is evident in unc-119 mutants. (E–H) Cell-specific rescue of unc-119 in C. elegans restores GPA-13 and ODR-3 localization. The unc-119 gene fused with GFP was driven by the gpa-13 promoter in ADF, ASH and AWC in unc-119 mutants. The transgenic (E,G) and unc-119 mutant control (F,H) were labeled with GPA-13 (E,F) or ODR-3 antibodies (G,H).