Figure 5. Hebp1 demonstrates perimitochondrial localization in neurons.

(A) Brain fractionation was performed as described before (Huttner et al., 1983). Hebp1 was identified in crude mitochondria fraction (Mt). Fraction annotation: H – homogenate, S1 – supernatant 1, P1 – pellet 1, S2 – supernatant 2 (fraction of soluble proteins), P2 – pellet 2 (synaptosomes), Mt – mitochondria, LP1 – lysate pellet 1 (plasma membrane fraction of synaptosomes), LS1 – lysate supernatant 1 (soluble fraction of synaptosomes). 20 µg of each fraction were loaded on the gel, except for LS1 (6 µg). (B) Mitochondria were isolated from cultured hippocampal neurons as described previously (Wieckowski et al., 2009). Hebp1 was only detected in isolated mitochondria together with Cox4 while markers for endo-lysosomal, synaptic and plasma membrane compartments (Lamp1, tubulin, Stx1, Rab5, Rab6 and VAMP2) were exclusively present in the supernatant. (C) Localization analysis of mitochondria (mitotracker), Hebp1-EGFP and EGFP alone in cultured rat hippocampal neurons (DIV14). Hebp1 puncta is associated with mitochondria. Representative line scans (golden lines in the inserts; location of the numbers correspond to the starting point of each analysis) were traced for EGFP control (D) and Hebp1-EGFP (E–F). Line scan analyses indicate that at least some of the Hebp1-EGFP puncta appear to be contacting mitochondria (E–F). Scale bar is 10 µm. All data shown are representative of results obtained from three independent experiments.