Abstract:
: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).
journal_name
FEBS Lettjournal_title
FEBS lettersauthors
Prescott M,Lourbakos A,Bateson M,Boyle G,Nagley P,Devenish RJdoi
10.1016/s0014-5793(97)00670-4subject
Has Abstractpub_date
1997-07-07 00:00:00pages
97-101issue
1eissn
0014-5793issn
1873-3468pii
S0014-5793(97)00670-4journal_volume
411pub_type
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