The yeast mitochondrial citrate transport protein: Determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecut ive residues within the fourth transmembrane domain (TMDIV). This domain ha s been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essentia l for CTP function and (ii) the existence of a transmembrane aqueous surfac e within this domain which likely corresponds to a portion of the citrate t ranslocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O-2 reveal an alpha-helical conformation along the sequence. The accessibilities to N iEDDA are out of phase with accessibilites to O-2, indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed a long the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the co ntext of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper repo rts the first use of site-directed spin labeling to study mitochondrial tra nsporter structure.