Fig. 2.

Endogenous succination does not alter tubulin polymerization in the brainstem of WT and Ndufs4 KO mice. A, BS samples were homogenized and centrifuged to obtain a cleared supernatant (Sup) as described under “Experimental Procedures.” When Sup was subjected to polymerization in the presence of taxol and high speed centrifugation, succinated tubulin (2SC panels) appeared in the microtubule pellet (Mt) but not in the remaining supernatant (SMt). Tubulin subunits (α-tubulin and β-tubulin panels) followed exactly the same pattern of distribution, indicating that endogenous succination of tubulin did not impair polymerization. Duplicate samples are shown for each genotype in this experiment; the same results were obtained in another experiment using a different set of samples. B, Total 2SC content (in mmol/mol Lys) was determined in Mt and SMt fractions after tubulin polymerization of BS samples by GC-MS/MS as described under “Experimental Procedures.” The distribution of succinated proteins favors the Mt fractions, especially in the BS of WT mice, whereas the BS of KO mice show a trend to increased total succination in the SMt fractions. Results are shown as the average of duplicate samples. C, Protein succination in the ∼50 kDa region of BS extracts from WT and Ndufs4 KO mice. For 30 μg protein samples succination increases in the KO group (30 μg, 2SC panel), but a slightly lower MW band is present in all samples (30 μg, Coomassie panel). With a reduced protein load of 5 μg, succination increases for the lower MW band in the BS of KO mice, with no changes in tubulin succination (5 μg, 2SC panel). β-tubulin and Coomassie staining are included to show even loading of the lanes (5 μg, β-tubulin and Coomassie panels). D–E, Cys³⁷⁶α is a prominent site of succination in the BS of the mouse. Mt fractions were resolved by SDS/PAGE and the tubulin bands at ∼50 kDa were excised and digested with trypsin prior to LC-MS/MS analysis as detailed under “Experimental Procedures.” D, MS/MS sequencing showing succination of Cys³⁷⁶α (C^2SC) in the peptide AVCMLSNTTAIAEAWAR; the pyridylethylated version of Cys³⁷⁶α (C^PE) is shown in (E).