CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.
The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.
Lurin
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Institut des Sciences Végétales, UPR 40, CNRS, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France.
Güclü
J
J
Cheniclet
C
C
Carde
J P
JP
Barbier-Brygoo
H
H
Maurel
C
C
D000906:Antibodies; D002458:Cell Fractionation / Q000379:methods; D018118:Chloride Channels / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000737:chemistry* / Q000648:ultrastructure; D008928:Mitochondria / Q000737:chemistry* / Q000648:ultrastructure; D010940:Plant Proteins / Q000032:analysis*; D010947:Plants, Toxic; D011994:Recombinant Proteins; D014026:Nicotiana / Q000737:chemistry* / Q000648:ultrastructure
D000906:Antibodies; C411344:CLC-Nt1 protein, Nicotiana tabacum; D018118:Chloride Channels; D010940:Plant Proteins; D011994:Recombinant Proteins