Abstract
The topography of the subunits of the membrane sector F0 of the ATP synthase complex in the bovine mitochondrial inner membrane was studied with the help of subunit-specific antibodies raised to the F0 subunits b, d, 6, F6, A6L, OSCP (oligomycin-sensitivity-conferring protein), and N,N' -dicyclohexylcarbodiimide (DCCD)-binding proteolipid and to the ATPase inhibitor protein (IF1) as an internal control. Exposure of F0 subunits in inverted and right-side-out inner membranes was investigated by direct antibody binding as well as by susceptibility of these subunits to degradation by various proteases as monitored by gel electrophoresis of the membrane digests and immunoblotting with the subunit-specific antibodies. Results show that subunits b, d, F6, A6L (including its C-terminal end) and OSCP were exposed on the matrix side. Sufficient masses of these subunits to recognize antibodies or undergo proteolysis were not exposed on the cytosolic side. This was also the case for subunit 6 and the DCCD-binding proteolipid on either side of the inner membrane. Quantitative immunoblotting in which bound radio-activity from [125I]protein A was employed to estimate the concentration of an antigen in a sample allowed the determination of the stoichiometry of several F0 subunits and IF1 relative to F1-ATPase. Results showed that per mol of F1 there are in bovine heart mitochondria 1 mol each of d, OSCP, and IF1, and 2 mol each of b and F6. Subunit 6 and the DCCD-binding proteolipid could not be quantitated, because the former transferred poorly to nitrocellulose and the latter's antibody did not bind [125I]protein A.
Highlights
From the $Divisionof Biochemistry, Department of Molecular and Experimental Medicine, Research Instituteof Scripps Clinic, La Jolla, California 92037 and the §Medical Genetics Division, Children’s Hospital, Los Angeles, California 90027
The sector FOof the ATP synthase complex in the bovine most complicated structure is found in mammalian mitochonmitochondrial inner membrane was studied with the dria, where the composition and stoichiometry of subunits help of subunit-specific antibodies raised tohe Fo sub- have yet to be fully clarified
Results (4-lo),littleis known abouttheirmembranetopography, show thatsubunits b, d, F6, A6L and OSCP wereexposed on the matrix ATPsynthesis.In a recentpaper, we reported improved side
Summary
Either a 1:lOO or a 1:50 trypsin to protein ratio. The digestion was allowed to proceedfor 30 or 120 min a t 37 "C and terminated by addition of 10-fold excess of soybean trypsin inhibitor. The partial inter- access of IgG to subunitb in ASU particles.Experiments with action of high antibody concentration with mitochondria andantisera directed against subunit 6, A6L peptides, and the mitoplasts was probably due to the presence of fragmented DCCD-binding proteolipidyielded negative results with both particles in these preparations and withASU particles due to SMP and ASU particles, suggesting that reactive epitopes the presence of residual F1. Each panel contains three lanes demon- during preparation of SMP Whether this material is a depstrating the resultosf digestion of the indicated particles with radation product of subunit b (a likely possihilitv as it is not trypsin for 0 (left lane), (mid& lane), and 120 (right lanc) always present, see Fig. 4 ) or a separate polypeptide that is min.Theantiserumwithwhicheachset of nitrocellulose recognizedbyouranti-suhunithantiserumremainstobe membranes was blotted is shown on thetop left-hand corner investigated.
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