Porphyrin and metallated porphyrin levels in undifferentiated MEL cells.

<p>(A) Hemin levels from undifferentiated control (n = 3) and FECH overexpressing (n = 4) MEL cells.. (B) Levels of protoporphyrin IX, Zn-protoporphyrin IX and porphyrin precursors (8–4 COOH porphyrins) for undifferentiated control (n = 3) and FECH overexpressing (n = 4) MEL cells. * indicates a <i>p</i> value of ≤ 0.006.</p

Subcellular fractionation and protease protection.

<p>7.5 ug of mitochondrial protein was processed and loaded in each lane. HB refers to treatment with hypo-osmotic buffer (20 mM KCl) for mitoplast isolation, PK to proteinase K (1 mg/mL) treatment and SDS to sodium dodecyl sulfate (0.5% w/v) treatment for mitoplast lysis. Markers for each compartment include hsp60—matrix, vdac1—outer mitochondrial membrane, tim23—inner mitochondria membrane and cytc—intermembrane space.</p

Proposed model of Mitochondrial Heme Metabolism Protein Complex.

<p>Proteins in the complex are shown in the inner mitochondrial membrane and labeled with their roles in porphyrin, iron and heme homeostasis. Additional proteins which may be involved in bridging between protein partners are not shown. IMM refers to the inner mitochondrial membrane, OMM to the outer mitochondrial membrane and EN to the endosome.</p

Native PAGE and Western blot analysis of mitochondrial protein complexes.

<p>(A) Solubilized proteins complexes from mitochondrial preparations of differentiated MEL cells were separated by Native PAGE. (B) Regions of the Native PAGE gel were excised and proteins further separated by SDS-PAGE. Western blot of the SDS-PAGE was carried out for ppox and fech.</p

Graphical representation of affinity purification and MS analysis of FLAG-FECH (red), FLAG-FECH Variants (purple), FLAG-PPOX (orange) and FLAG-CPOX (blue).

<p>Each panel represents an identified mouse protein recovered with the bait human protein listed in the legend of panel A. Panels are as follows: (A)—fech, (B)—ppox, (C)—cpox and (D)—alas2, (E)—sucla2, (F)—abcb10 and (G)—abcb7. Number of unique peptides (x axis), % sequence coverage (y axis) and spectral counts (bubble size) for each was calculated using the maximal values obtained minus the maximal values observed in the control samples (empty vector). Size of bubbles represents the % of the total spectral counts identified for each mouse protein. The maximal spectral counts of each of the mouse proteins was fech = 971, ppox = 71, cpox = 256, alas-2 = 16, sucla2 = 16, abcb10 = 51 and abcb7 = 36. Note no mouse peptides for alas2, sucla2, or abcb10 were obtained using FLAG-CPOX as bait above that of empty vector.</p

Immunoblot from Affinity Purification of FLAG elutions.

<p>Each lane represents the FLAG elutions from the affinity purification using empty vector (lane 1), WT FECH (lane 2), M76H FECH variant (lane 3) and E343K FECH variant (lane 4). Blots were probed for abcb7, abcb10, sucla2, ppox and fech. The dashed line indicates non consecutive lanes on the same gel.</p