Entrapment by Immobilized Metal Ion Affinity Chromatography of Assembled Yeast Mitochondrial ATP Synthase Containing Individual Subunits Tagged with Hexahistidine

A novel fluorescent marker for assembled mitochondria ATP synthase of yeast

“…By contrast, polypeptides corresponding to mtATPase were not retrieved in eluates from Ni-NTA loaded with lysates of mitochondria isolated from cells of the control strain YRD15 (lane 3). A few mitochondrial polypeptides non-specifically absorbed to and eluted from Ni-NTA resin have been previously observed [5].…”

“…GFP was fused at the C-terminus of OSCP in anticipation of minimal interference with mitochondrial targeting and accurate proteolytic maturation of the OSCP moiety in mitochondria. We noted that OSCP can tolerate substantial additions at its C-terminus ( [5]; M. Prescott, unpublished observations). In addition, the gene cassette for GFP was modified to encode a C-terminal addition of hexahistidine.…”

“…As previously described [5] proteins were separated by SDS-PAGE and stained with silver or transferred to nitrocellulose and probed with rabbit polyclonal antisera against OSCP diluted 1/1000. GFP was similarly detected by probing with mouse monoclonal antiserum against GFP diluted 1/1000 (kindly provided by Dr. John Gill, Boehringer-Mannheim Biochemicals, Indianapolis, IN).…”

“…This modification was made to allow assembly of the fusion protein into mtATPase complexes to be readily assessed. The entire mtATPase complex containing a single subunit tagged at the C-terminus with hexahistidine can be rapidly and conveniently isolated under relatively mild conditions using metal ion chelate chromatography [5].…”

“…Use was made of the Ni-NTA chromatography technique to purify assembled mtATPase complexes [5] through selective adsorption of the hexahistidine tag attached to the fusion protein. Mitochondrial lysates of AL1 cells and cells of the control strain YRD15 were prepared and subjected to chromatography on Ni-NTA resin.…”