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 carrier in the inner mitochondrial membrane and in the membrane of reconstituted proteoliposomes has been investigated by enzymatic and immunochemical approaches using proteinase K and polyclonal site-directed antibodies, respectively. Two peptides corresponding to the amino acid sequences 2-12 (N-terminal peptide) and 303-314 (C-terminal peptide) have been synthesized and coupled to ovalbumin before being used to immunize 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 specifically with the corresponding peptides and with the isolated oxoglutarate carrier, whereas only anti-C-terminal antibodies immunodetected the carrier in mitochondrial lysates and reacted with the membrane-bound carrier in mitoplasts and in freeze-thawed mitochondria. This result indicated that the last 12 C-terminal amino acid residues of the oxoglutarate carrier protein are accessible from the cytosolic side of the inner mitochondrial membrane. Anti-C-terminal antibodies did not recognize the oxoglutarate carrier in reconstituted proteoliposomes unless the membrane was inverted, indicating that the carrier was inserted unidirectionally in proteoliposomes, with an orientation opposite that found in mitochondria. The immunological data were complemented by data from a limited proteolysis study performed on the membrane-bound oxoglutarate carrier in proteoliposomes, using proteinase K. Cleavage of the carrier caused a time-dependent inhibition of the oxoglutarate-oxoglutarate exchange activity of the reconstituted system. Four cleavage sites 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Bisaccia
F
F
Department of Pharmaco-Biology, University of Bari, Italy.
Capobianco
L
L
Brandolin
G
G
Palmieri
F
F
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D019286:Endopeptidase K; D004797:Enzyme-Linked Immunosorbent Assay; D007425:Intracellular Membranes / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000737:chemistry* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; C021723:oxoglutarate translocator; D012697:Serine Endopeptidases; D019286:Endopeptidase K