The mitochondrial oxoglutarate carrier: structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling.
The structural and dynamic features of the fourth transmembrane segment of the mitochondrial oxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance (EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutive residues from S184 to S201 which are believed to form the transmembrane segment IV were substituted individually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutants exhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined by EPR as a function of the incident microwave power in the presence and absence of two paramagnetic perturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. The periodicity of the sequence-specific variation in the spin-label mobility and the O(2) accessibility parameters unambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier as an alpha-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicating that the helix is amphipatic, with the hydrophilic face containing the residues found to be important for transport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed, as indicated by the values of normalized mobility, which also suggest that the conformational changes occurring during transport probably involve the N-terminal region of the helix.
INFM and Department of Biology, University of Rome Tor Vergata, via della Ricerca Scientifica snc, 00133 Rome, Italy.
Blasco
B
Morozzo della Rocca
Graziantonio
G
Lauria
Francesca
F
Venerini
Luigi
L
Palmieri
Francesca
F
Polizio
Loredana
L
Capobianco
Valentina
V
Stipani
Jens
J
Pedersen
Anna Rita
AR
Cappello
Alessandro
A
Desideri
Ferdinando
F
Palmieri
D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology*; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007656:Ketoglutaric Acids / Q000378:metabolism; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010070:Oxalates / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D021381:Protein Transport; D011510:Proteolipids; D011994:Recombinant Proteins / Q000378:metabolism; D013113:Spin Labels
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; D010070:Oxalates; D011510:Proteolipids; D011994:Recombinant Proteins; D013113:Spin Labels; C021723:oxoglutarate translocator; C020485:proteoliposomes; D003545:Cysteine; D010100:Oxygen