A novel fluorescent marker for assembled mitochondria ATP synthase of yeast. OSCP subunit fused to green fluorescent protein is assembled into the complex in vivo.
We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C-terminus to the green fluorescent protein (GFP) of Aequorea victoria. The relevant fusion OSCP-GFP-h6 additionally contains a hexahistidine tag at the C-terminus. Expression of OSCP-GFP-h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non-fermentable substrate, ethanol. Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP-GFP-h6. Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP-GFP-h6 identified by its mobility on SDS-PAGE and immunoreactivity to anti-OSCP and anti-GFP antibodies. The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP. In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET).
Prescott
M
M
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Lourbakos
A
A
Bateson
M
M
Boyle
G
G
Nagley
P
P
Devenish
R J
RJ
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D000431:Ethanol / Q000494:pharmacology; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D025261:Mitochondrial Proton-Translocating ATPases; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure
D002352:Carrier Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000431:Ethanol; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein