Figure 6.

Mitochondrial targeted PXN protein is functional. A, Fusion of the mitochondrial target signal (mt) allows the localization of PXN to yeast mitochondria. The mt-PXN-EYFP fusion protein was constitutively expressed in BY4741 yeast cells (pMSU388). Localization study was analyzed by fluorescence microscopy. To visualize mitochondria, yeast cells were stained with MitoTracker Orange. Scale bar = 5 μm. B, Gal-inducible expression of mt-PXN-His (39.1 kD) in the FGY217 yeast strain. Left, Coomassie-stained SDS-PAGE gel. Right, Immunoblot blot treated with His-tag antibody. Lane 1, Yeast membrane proteins isolated from yeast cells containing empty vector pNL33; lane 2, yeast membrane proteins isolated from yeast cells expressing mt-PXN-His (pMSU237); M, protein marker. C, Time-dependent uptake of radioactively labeled [α-³²P]-NAD (0.2 mm) was measured in liposomes reconstituted with total yeast membranes containing mt-PXN-His. The proteoliposomes were preloaded internally with 10 mm NAD (black symbols) or lack NAD as counter-exchange substrate (white symbols). Graphs represent the arithmetic mean ± se of three technical replicates.