CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes withmitochondrial membrane markers

The voltage-dependent chloride channel (CLC) family of membrane proteins ha s cognates in animals, yeast, bacteria and plants, and chloride-channel act ivity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localiz ation of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were r aised against an N-terminal polypeptide of CLC-Nt1. These antibodies were u sed to probe membrane proteins prepared by various cell-fractionation metho ds. These included aqueous two-phase partitioning (for plasma membranes), f ree-flow electrophoresis (for vacuolar and plasma membranes), intact vacuol e isolation, Percoll-gradient centrifugation (for plastids and mitochondria ) and stepped, linear, sucrose-density-gradient centrifugation (for mitocho ndria). Each purified membrane fraction was characterized with specific mar ker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma memb ranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In c ontrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and N AD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('I MAC') described previously in animal and plant mitochondria.