Transgenic ^Δ1–83PhLP in the rod photoreceptor forms a tertiary complex with the chaperonin CCT and the Gβ₁ subunit resulting in loss of visual function. A, Transgene utilized for the expression of FLAG-tagged ^Δ1–83PhLP in mouse photoreceptors. B, Western blot analyses of FLAG-agarose pull-down products in the retinal extracts of transgene-negative Tg(-) and transgene-positive Tg(+) mice at P10, detected with antibodies against FLAG, TCP-1α, TCP-1β, TCP-1γ, TCP-1ε, Gβ₁, and Gγ₁, as indicated. C, Model of PhLP-dependent delivery and release of Gβ₁ from the chaperonin CCT that shows formation of a stable complex between ^Δ1–83PhLP, CCT, and Gβ₁. D, Typical electroretinography responses at P25, evoked by a −10 dB UTAS-E4000 flash (0.25 candela per second per square meter). Traces were obtained using a 300 Hz high-cut filter and represent the averaging of 5 responses.

Quantitative proteomics of the CCT-deficiency in retina. A, Approach for the quantitative analysis of the changes in the retina proteome induced in the ^Δ1–83PhLP transgenic model. Total protein was extracted in parallel from the retinas of wild-type (Tg-) and ^Δ1–83PhLP transgenic (Tg+) mice, and samples were reduced, alkylated and digested with trypsin. Peptides were differentially labeled with iTRAQ® reagents as indicated, i.e. iTRAQ®114 and 115 (●) were used for Tg(-), iTRAQ®116 and 117 (♦) for Tg(+) samples. Samples were combined, resolved by two-dimensional high pressure liquid chromatography, continuously spotted on a MALDI target upon mixing with matrix solution, and analyzed by tandem mass spectrometry using an ABI 4800 MALDI TOF/TOF analyzer. The change in protein abundance results in a similar change in iTRAQ® reporter ion intensities, allowing for the relative quantification of protein expression. Alternatively, total protein extracts from Tg(-) and Tg(+) retinas were differentially labeled with CyDyes as shown, combined and resolved by a two-dimensional difference gel electrophoresis (two-dimensional DIGE). Gels were scanned and spots showing differences in the amounts of protein, as detected by in-gel and cross-gel analyses of the images, were robotically picked, reduced, alkylated and digested with trypsin. The resulting peptide mixture was analyzed by tandem mass-spectrometry using an ABI 4700 MALDI TOF/TOF analyzer. B, Representative MS/MS fragmentation spectra of peptides assigned to up- and downregulated proteins. Continuous aa series from 438 to 444 of heat shock protein 1-α (Hsp1a) and from 89 to 110 of retinal-S-antigen (Arr) sequences. Inserts: high resolution graphs of iTRAQ® reporter ion region showing increased (Hsp1a) or decreased (Arr) abundance of the corresponding peptides in Tg(+) samples. All graphs were exported as ASCII files, and peaks were labeled in the GraphPad Prism (La Jolla, CA). Abbreviations are: aa, amino acids; cps, counts per second; Rep, iTRAQ® reporter ions. C, Distribution analysis of significantly changed proteins. For all proteins found to be significantly and consistently changed in iTRAQ® experiments, the total number of proteins was plotted as a function of the average linear fold change with an increment of 0.05. The dashed line represents the verge between downregulated (fold change <1) and up-regulated (fold change >1) proteins.

^Δ1–83PhLP expression induces a massive down-regulation in critical components of the phototransduction cascade. A, Schematic of the core reactions in the phototransduction cascade. Proteins within this pathway found to be down-regulated are shown in green. Connecting line indicates direct binding, T-shaped lines indicate inhibition, thick arrows indicate activation, and thin arrows represent the direction of the chemical reaction. Phosphorylation is indicated as P. Protein complexes are outlined by dashed line. Rh, rhodopsin; ARR, arrestin; PDC, phosducin; PDE, phosphodiesterase; RGS9, regulator of G protein signaling 9; R9AP, RGS9 anchoring protein; GRK, G protein coupled receptor kinase; cGGC, cGMP gated channels. B, Validation of proteins essential for photoreceptor viability by quantitative Western blotting. Representative Western blots showing the levels of the indicated proteins in the whole retina extracts of Tg(-) and Tg(+) littermates at P10. Quantitative Western blotting was conducted as outlined in the Experimental Procedures. Bar graphs below each set of blots show the change of each protein level (1–10), expressed as a negative ratio of the specific band fluorescence in Tg(-) and Tg(+) samples. A specific band corresponding to the transducin α subunit was undetectable in the Tg(+) sample. Data were averaged using at least 3 littermates of each genotype and a Student t test unpaired, errors are S.E., p < 0.01.

The effect of ^Δ1–83PhLP expression on photoreceptor structure and viability. A, Light microscopy images illustrating the outer retina anatomy in transgene-negative Tg(-) and transgene-positive Tg(+) mice of the indicated postnatal ages. B, Ultrastructure of the photoreceptor outer segment at P10. Inserts show photoreceptor plasma membrane outlined with black. Abbreviations: ONL, outer nuclear layers; OS, photoreceptor outer segments; CC, connecting cilium.

Functional analysis of the proteome changes triggered by the CCT deficiency. A, Categorical analysis based on the molecular function of up- and down-regulated proteins performed using GenePept and KEGG BRITE databases. Category of unknown/miscellaneous proteins is not shown. B, Network analysis of the functional pathways identified by an Ingenuity Pathway Analysis. Proteins were grouped and connected into pathways on the basis of physiological function. These pathways were concomitantly overlaid over all proteins, and proteins not integrated into the network were trimmed. Red shapes indicate up-regulated proteins, green, down-regulated. Triangles represent molecules incorporated in four or more pathways.