Transducin gamma-subunit sets expression levels of alpha- and beta-subunits and is crucial for rod viability

Abstract

Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the alpha- and betagamma-subunits. Here we demonstrate that the knock-out of transducin gamma-subunit leads to a major downregulation of both alpha- and beta-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining alpha- and beta-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the alpha-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the gamma-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin beta-subunit without its constitutive gamma-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.

Figure 1.

Knock-out strategy and genotyping of the Gγ₁ knock-out mouse. A, Structures of the wild-type Gngt1 gene locus containing exons 1 and 2 and the targeting vector. Dark boxes represent regions of the Gγ₁ coding sequence (amino acid residues 17–44), which was replaced with a 6.9 kb IRES-lacZ reporter and neomycin-resistance cassette (IRES-lacZ-neo). B, Genotyping of Gngt1 knock-out mice by multiplex PCR. We used a mixture of three primers: a (5-TGC TCA CTC TCC TCC ATC TTC ACA C-3), b (5-CTG GAA TCC CCT TCA CTA GAG GGT C-3), and c (5-GAC GAG TTC TTC TGA GGG GAT CGA TC-3), which amplify the 412 bp product from the Gngt1 gene (a and b) and/or the 617 bp product from the knock-out allele containing a fragment of the neomycin resistance cassette (b and c). Genomic DNA was isolated from the mouse tail tips using the DNeasy Tissue kit (QIAGEN).

Figure 2.

Comparative analysis of retina morphology in Gγ₁ knock-out (−/−), heterozygous (+/−), and wild-type littermates (+/+) and Gα_t/Gγ₁ double knock-out (KO) and phosducin (Pdc)/Gγ₁ double knock-out mice. Animals were killed at indicated ages, retinas were embedded in plastic, and 1 μm cross sections were stained by toluidine blue and analyzed using a Nikon (Tokyo, Japan) Eclipse 90i microscope.

Figure 3.

Transmission electron microscopy of rod outer segments from 0.065 μm retina cross sections from the 1-month-old Gγ₁ knock-out mouse and its heterozygote and wild-type littermates. The pictures were taken at 2500× magnification.

Figure 4.

The expression of transducin and other major photoreceptor proteins in the retinas of 31- to 33-d-old Gγ₁ knock-out mice. A, Western blots of proteins in the retina lysates containing 5 pmol of rhodopsin. Each determination was repeated for at least three pairs of wild-type and knock-out animals. B, Quantification of transducin subunit amounts in whole retinas from Gγ₁ knock-out, heterozygote, and wild-type littermates. Retina lysate aliquots containing indicated amounts of rhodopsin were separated by SDS-PAGE along with 0.05, 0.1, 0.2, 0.3, and 0.4 pmol of transducin standards and immunoblotted using antibodies against each subunit. The examples of calibration curves for each subunit are shown below the blots. The results from multiple experiments are summarized in Table 1. C, The distribution of Gβ₁ in 20 μm serial tangential sections throughout the entire light-adapted retina of wild-type and Gγ₁ knock-out mice was analyzed by Western blotting. Each section was solubilized in either 40 (knock-out) or 60 (wild-type) μl of SDS-PAGE sample buffer, and 10 μl aliquots were subjected to electrophoresis. The final blots were scanned at the excitation laser intensity optimized for attaining signal linearity in each case. The representative cross sections of each retina type are shown above the corresponding Western blot panes; note that the photoreceptor layer in knock-out retinas is approximately one section thinner than in wild type because of ongoing degeneration. D, Transcript levels of transducin subunits in Gγ₁ knock-out, heterozygote, and wild-type littermates. Quantitative RT-PCR of each transcript was conducted for two animals of each type as described in Materials and Methods. The relative mRNA expression level in each sample was normalized to the fluorescence of GAPDH and shown as the fraction of wild type.

Figure 5.

The distribution of Gα_t and Gβ₁ in 10-μm-thick tangential sections of the photoreceptor layer from Gγ₁ knock-out and wild-type mice. Dark-adapted animals were anesthetized and either kept in the dark or exposed to 30 min of illumination, bleaching at least 80% rhodopsin by the end of the experiment. Their retinas were extracted and sections were obtained as by Lobanova et al. (2007). Each section from knock-out retinas was solubilized in 30 μl and from wild type in 100 μl of SDS-PAGE buffer, and 12 μl aliquots were used for Western blotting. A small 1 μl aliquot from each sample was probed for the presence of rhodopsin (Rho), serving as the rod outer segment marker. The data are taken from one of at least three independent experiments. A schematic drawing of the rod cell is shown between the panels with subcellular compartments abbreviated as follows: OS, outer segment; IS, inner segment; N, nucleus; ST, synaptic terminal.

Figure 6.

The distribution of Gβ₁ and Gγ₁ in 5-μm-thick serial tangential sections of the photoreceptor layer in dark-adapted (A) or light-adapted (B) Gα_t knock-out (KO) mice and in dark-adapted double Gα_t/phosducin (Pdc) knock-out mouse (C). The experiments were performed as described in Figure 5 legend (wild type), except that the much higher Gβ₁γ₁ content in these animals enabled us to analyze proteins in thinner 5 μm sections.

Figure 7.

ERG analysis of light responses in Gγ₁ knock-out mice. A, Representative ERG recordings from 31- to 33-d-old wild-type, Gγ₁ knock-out, and Gα_t knock-out mice evoked by white light flashes of increasing intensities indicated to the right of the traces as the log intensity measured in cd · s/m². B, The dependencies of a- and b-wave amplitudes in each animal type on the flash intensity. The curve fitting was performed based on a modified hyperbolic function according to Fulton and Rushton (1978): where A_max is the maximal amplitude, I is the flash intensity, n is the Hill coefficient, and I_h is the half-saturating light intensity. In wild-type mice, the first term represents the contribution from rods, and the second represents the contribution from cones (observed in bright light). The parameters of the fits are summarized in Table 3.

Figure 8.

Light responses of individual wild-type and Gγ₁ knock-out rods. A, Families of flash responses from representative wild-type and Gγ₁ knock-out rods. Flash strengths ranged from 6.82 to 2937 photons/μm² for WT and from 872 to 73698 photons/μm² for Gγ₁ knock-outs. B, Normalized response amplitudes as a function of flash strength for the Gγ₁ knock-out rod shown in A (open circles) and a rod from C57BL/6 wild-type mouse (filled squares). Points were fitted by saturating exponential functions. Dark currents were 16.5 pA (wild type) and 12.3 pA (Gγ₁ knock-out).

Figure 9.

Cellular dark noise variance in Gγ₁ knock-out rods. A, Representative current recordings from WT and Gγ₁ knock-out rods in darkness (bottom) and in saturating light that closed all of the cGMP-gated channels (top). B, Difference power spectra (dark − light) for each rod revealed a marked decrease in cellular dark noise in the lower frequency ranges, consistent with reduced transduction noise. Open circles, Gγ₁ knock-out rods; filled circles, wild-type rods. Dark currents were 12.6 pA (WT) and 10.4 pA (Gγ₁ knock-out).