Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.
Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.
Dentler
W L
WL
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045.
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002923:Cilia / Q000737:chemistry*; D003208:Concanavalin A; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000032:analysis; D008870:Microtubules / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D015800:Protozoan Proteins / Q000032:analysis*; D013388:Succinimides; D016808:Tetrahymena thermophila / Q000737:chemistry*
D008565:Membrane Proteins; D015800:Protozoan Proteins; D013388:Succinimides; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D003208:Concanavalin A; D011092:Polyethylene Glycols; D001710:Biotin; D017830:Octoxynol; C010615:Nonidet P-40