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. 2014 May 2;289(25):17427–17444. doi: 10.1074/jbc.M114.568949

FIGURE 5.

FIGURE 5.

Stoichiometry of Ribbon proteins. a, stoichiometry of tektins. Ribbons were extracted once with Sarkosyl-urea, leaving a residual amount of associated tubulin but minimizing the loss of tektin C, resolved by SDS-PAGE, and stained quantitatively with Serva Blue. Gel lanes were scanned, and the stain intensities of α-tubulin and tektins A, B, and C measured. Because β-tubulin migrates closely with tektin B (Fig. 4a, lane 2) and because the moles of α-tubulin must equal the moles of β-tubulin, the stain intensity of α-tubulin was subtracted from the intensity of the (tektin B + β-tubulin) peak to determine the amount of tektin B alone. The intensities of tektins A, B, and C were divided by their masses (53, 51, and 47 kDa, respectively) to give intensity/kDa and normalized to tektin A. The molar ratio of tektins A/B/C was thus determined to be 1:1:1. Dashed lines indicate the separation of the intensities of the individual polypeptides, and the dotted lines indicate the registration of the respective polypeptides in b. b, stoichiometry of Ribbon proteins. DMTs were extracted once with Sarkosyl (to minimize sample loss) and analyzed as described for tektin filaments above. Dashed lines indicate the separation of the intensities of the individual polypeptides. The stoichiometry of the Ribbon proteins was thus calculated and reported in Table 1. c, the number of Ribbons per axoneme was estimated as follows. Reference lanes were loaded with the amount of axonemal tubulin calculated for one, two, and three Ribbons (of three tubulin PFs) per DMT (i.e. 11.6, 23.2, and 34.8%, respectively), against which the experimental Ribbon sample was compared. The α-tubulin region was measured (bracket, dashed lines) and plotted as % of axonemal α-tubulin versus the integrated (stain) intensity of α-tubulin, ■. The experimentally obtained Ribbon α-tubulin (+) corresponds to ∼14% of the axonemal α-tubulin, very close to the amount (11.6%) expected for one Ribbon of three tubulin PFs per DMT. The value 14.1% is probably artificially high and closer to the theoretical 11.6%, because (i) central pair-MTs are less stable than DMTs and therefore some central pair-tubulin is lost during the isolation of axonemes, and (ii) because a small percentage (<5%) of the once-extracted Ribbons contains four PFs and not three.