Figure 4: Protein N-myristoylation is required for AMPK association with mitochondria and mitochondrial removal. | Nature Communications

Figure 4: Protein N-myristoylation is required for AMPK association with mitochondria and mitochondrial removal.

From: Myristoylation confers noncanonical AMPK functions in autophagy selectivity and mitochondrial surveillance

Figure 4

(a) Detection of myristoylated AMPKβ1 (Myr-β1) co-precipitated with GFP-AMPKα2 following metabolic labelling of H23 cells with azidomyristate and in vitro conjugation with biotin alkyne. Input protein levels were determined by immunoblotting. Note that biotin-conjugated azidomyristoylated AMPKβ1 migrated relatively slowly. (b) Western analyses of myristoylated AMPKβ1 (Myr-β1) levels in the whole-cell lysate (WCL) and mitochondrial fraction (Mito) of H23 cells treated with or without CCCP following azidomyristic acid labelling and click chemistry. WCL without click conjugation was loaded as controls. Bafilomycin (Baf) was used to prevent autophagy/mitophagy-mediated protein degradation. LDHB was blotted for loading control. The bottom panel shows overall levels of protein myristoylation (streptavidin blot) in the mitochondria. ImageJ quantification was performed to assess the relative abundance of the proteins (fold changes). (c) Detection of overall levels of protein myristoylation in mitochondria (Mito) and cytosol (sup) following metabolic labelling and click chemistry in H23 cells treated with (+) or without (−) 2-hydroxy myristic acid (2HMA) and CCCP. (d) Western analyses of protein levels from H23 cells treated without (−) or with (+) CCCP (30 μM) for 4 h in the presence (+) or absence (−) of 2HMA (0.5 mM; added 20 h before CCCP). WCL, whole-cell lysate; MF, membrane fraction extracted using the Cell Fractionation Kit, HT (Ab109718) from Abcam (Cambridge, MA, USA). Note that AMPK complex was not enriched in the membrane fraction containing the plasma membrane and other organelles in addition to the mitochondria. ** denotes absence of vimentin in membrane fraction and presence of a faster migration band of non-characterized origin in 2HMA-treated cells. (e) Representative transmission electron microscopy micrographs of H23 cells treated with (+) or without (−) 2HMA (0.5 mM) for 24 h before brief exposure to CCCP (15 μM) in the presence of bafilomycin A (100 nM). Red arrows, autophagosomes; blue triangles, mitochondria; scale bar, 500 nM.

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