TRANSMEMBRANE TOPOGRAPHY OF THE MITOCHONDRIAL OXOGLUTARATE CARRIER ASSESSED BY PEPTIDE-SPECIFIC ANTIBODIES AND ENZYMATIC CLEAVAGE

The folding of the peptide chain of the bovine heart oxoglutarate carr ier in the inner mitochondrial membrane and in the membrane of reconst ituted proteoliposomes has been investigated by enzymatic and immunoch emical approaches using proteinase K and polyclonal site-directed anti bodies, respectively. Two peptides corresponding to the amino acid seq uences 2-12 (N-terminal peptide) and 303-314 (C-terminal peptide) have been synthesized and coupled to ovalbumin before being used to immuni ze rabbits. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and by Western blot analysis . Both anti-N-terminal and anti-C-terminal antibodies reacted specific ally with the corresponding peptides and with the isolated oxoglutarat e carrier, whereas only anti-C-terminal antibodies immunodetected the carrier in mitochondrial lysates and reacted with the membrane-bound c arrier in mitoplasts and in freeze-thawed mitochondria. This result in dicated that the last 12 C-terminal amino acid residues of the oxoglut arate carrier protein are accessible from the cytosolic side of the in ner mitochondrial membrane. Anti-C-terminal antibodies did not recogni ze the oxoglutarate carrier in reconstituted proteoliposomes unless th e membrane was inverted, indicating that the carrier was inserted unid irectionally in proteoliposomes, with an orientation opposite that fou nd in mitochondria. The immunological data were complemented by data f rom a limited proteolysis study performed on the membrane-bound oxoglu tarate carrier in proteoliposomes, using proteinase K. Cleavage of the carrier caused a time-dependent inhibition of the oxoglutarate-oxoglu tarate exchange activity of the reconstituted system. Four cleavage si tes were identified, between Val-39 and Gln-40, between Tyr-61 and Lys -62, between Phe-169 and Arg-170, and between Arg-182 and Gly-183. The se sites were assigned to the external side of the liposomal membrane and therefore to the matrix side of the inner mitochondrial membrane. The presence of three additional cleavage sites, located between Ala-5 and Ser-6, between Ser-22 and Val-23, and between Thr-103 and Val-104 , was demonstrated in proteolysis experiments with inside-out proteoli posomes. It was concluded that the latter three sites are exposed to t he internal side of the liposomal membrane and oriented toward the cyt osol in intact mitochondria. These results are consistent with an arra ngement of the peptide chain of the oxoglutarate carrier monomer into an even number of transmembrane segments, with the N- and C-terminal r egions protruding into the cytosol.