The Journal of Neuroscience, May 16, 2007, ():
Subunit Dissociation and Diffusion Determine the Subcellular Localization of Rod and Cone Transducins
J. Neurosci. Rosenzweig et al.
27: 5484
Supplemental Data
Files in this Data Supplement:
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Supplemental text
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Figure S1. MS and MS/MS spectra of N-terminal fatty acyl tryptic peptides derived from rod and cone G? subunits.
The mass-spec spectra shown in this figure correspond to the peaks observed in the ion chromatograms (Fig. 5A).
A-D. The molecular ions of N-terminal C14:0-, C14:1-, C14:2-, and C12:0-rod transducin peptides and their corresponding CID MS/MS spectra, which confirmed the identities of the peptides in the HPLC-MS profiles. E, F. The molecular ion of N-terminal C14:0-cone transducin peptide and its CID MS/MS spectrum. The peptide was obtained from Nrl-/- and Gnat2+,Gnat1-/- retinas. Due to the application of a different HPLC gradient program, C14:0-cone transducin peptide gave a different retention time in (E) and (F), while their assignments were confirmed by the MS/MS spectrum.
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Figure S2. Comparison of the amino aid sequences of rod and cone G? subunits.
Alignment of N-terminal amino acid sequences of mouse rod and cone alpha subunits with G?i and G?o sequences reveals an apparent deletion of four amino acids in rod G?t.
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Figure S3. The rate of protein diffusion in mouse cones.
FRAP experiments were performed on retinas of GFP-expressing (mosaic) mice, as described in (Nair et al., 2005). Live retinas were dissected, embedded in low-melt agarose, sliced on a vibratome and imaged on Zeiss LSM-500 laser confocal microscope. GFP-expressing cones were identified by DIC according to their morphology (relatively short OS compared to the essentially uniform rod OS), which located somewhat “below” the layer of rod OS. The number of such cells was small in each specimen. The selected cells were subjected to FRAP as follows: (i) an image of the cell prior photobleaching was taken (Pre-bleach) (ii) an area including the OS shown as the box in the panels was irradiated with maximal laser intensity, (iii) images are taken, every 30 seconds, (iv) the fluorescence intensity within the selected box was measured and normalized to the overall bleaching of the visual field, which occurs due to the repeated laser scans. The normalized intensity of green fluorescence recovery was plotted against time. The graph shows mean +/- SD from two independent experiments.
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Figure S4. Model of the mechanism of light-induced transducin dispersion.
In the dark, the G? subunit of rod transducin (blue) is associated with G?1?1 (light green and brown), and the heterotrimer is associated with the OS disc membranes. Similarly, cone G?t (green) is associated with G?3?8, (red and violet) and bound to cone OS membranes. Upon stimulation by light, both cone and rod G? bind GTP and undergo a conformational change to the active state (symbolized by a lighter color). For rod transducin this causes the subunits to dissociate, release from the membrane and disperse throughout the cytosol of the entire rod cell. Cone transducin G?t-GTP does not physically dissociate from G??, so the heterotrimer stays attached to the membrane sequestered in the OS.
