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. 2020 Jun 18;21(8):e48882. doi: 10.15252/embr.201948882

Figure 4. Mitochondrial proteins locally synthesized in the synapse are incorporated into respiratory chain complexes.

Figure 4

  • A
    Synaptoneurosomes were NMDA‐R‐stimulated and incubated with radioactive 35S‐methionine/cysteine mix for 1 h. Samples were separated using blue‐native electrophoresis (BN‐PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN‐PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.
  • B, C
    To block protein import and incorporation of newly synthesized proteins into respiratory complexes, the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m‐chlorophenyl hydrazone (10 μM CCCP) (B) or VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, and 8 μM antimycin) (C). Additionally, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3 mM) (C). Protein complexes were separated on BN‐PAGE and visualized by Coomassie staining and autoradiography. One hour after the stimulation, significant increase in 35S‐labeled proteins was observed (B, n = 4 biological replicates, *P < 0.0145; C, n = 5 biological replicates, **P = 0.0023; repeated‐measures one‐way ANOVA, post hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S‐methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n = 4 biological replicates, *P = 0.02, repeated‐measures one‐way ANOVA, post hoc Sidak's multiple comparisons test) or VOA (C; n = 5 biological replicates, **P = 0.0044, repeated‐measures one‐way ANOVA, post hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S‐methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n = 5 biological replicates, *P = 0.0236, repeated‐measures one‐way ANOVA, post hoc Sidak's multiple comparisons test). Error bars indicate SEM.

Source data are available online for this figure.