Fractionation of Tetrahymena Ciliary Membranes with Triton X-U4 and the Identification of a Ciliary Membrane ATPase

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble ct and 13 tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg ++ or Ca ++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane. C ILIARY and flagellar membranes are associated with a number of metabolic and structural processes, including olfaction (Chen et al., 1986), Ca ++ ion regulation (Travis and Nelson, 1986), regulation of ciliary motility (Gustin and Nelson, 1987; Dentler et al., 1980), microtubule assembly (Dentler, 1980a), and gliding (Bloodgood, 1977). Each of these properties require membrane proteins to be properly oriented and localized along the ciliary and flagellar shaft. Little is known about the structure or organization of ciliary membrane proteins but Stephens et al. (1987) proposed that a detergent-resistant membrane skeleton, composed partly of "membrane tubulin", is associated with the cytoplasmic face of scallop gill ciliary membranes and that this skeleton may provide structural rigidity to the membrane as well as attachment sites for the microtubule-membrane bridge proteins (Dentler et al., 1980). Ciliary membranes and detergent-soluble "membrane + matrix" fractions are composed of 25-50 (or more) different polypeptides, as resolved on one-dimensional SDS-PAGE (Chen et al., 1986; Dentler, 1980b; Adoutte et al., 1980). To understand the functions of these proteins, it is necessary to identify the locations of these proteins in the membrane and identify proteins that may be associated with the membrane skeleton, if such a skeleton exists in all cilia. In this study, Tetrahymena ciliary membranes were fractionated with Triton X-114 to separate and identify integral membrane proteins (Bordier, 1981; Chen et al., 1986; Stephens, 1985a; Pryde and Phillips, 1986) and to determine if the major detergent-soluble proteins, previously identified as tubulins (Dentler, 1980b), are integral membrane proteins. The resuits show that the major Tetrahymena thermophila membrane proteins migrate at 50 and 58 kD, partition into the Triton X-114 detergent phase and are glycosylated and phosphorylated in vivo. The detergent-soluble Tetrahymena ciliary tubulin partitions into the aqueous phase and is unlikely to be an integral membrane protein. Since ciliary and flagellar membrane ATPases have been described in a number of organisms (see Dentler, 1981; Travis and Nelson, 1986), the Triton X-114 aqueous and detergent phase fractions were examined for ATPase activity. The results show nearly 20% of the total ciliary ATPase activity is released by Triton X-114, most of which partitions into the detergent phase. The membrane ATPase is insensitive to vanadate and ouabain and requires divalent cations. The function of the ATPase is unknown but it could be a membrane pump or be related to the movements of ciliary surface proteins (Williams et al., 1985; Bloodgood, 1987). Materials and Methods Isolation of Cilia and Triton X-114 Fractions Cilia were isolated from Tetrahymena thermophila, strain B-255 (from E. Orias, University of California, Santa Barbara) using dibucaine as previousty described (Suprenant and Dentler, 1988). Cells were grown in 2% proteose peptone (Difco Laboratories Inc., Detroit, MI), 0.1 mM FeCI3, and 0.0025 % penicillin-streptomycin in 2.8-liter fernbach flasks at room temperature on an orbital shaking table. For most experiments, 3 liters of cells grown 1o late log phase, harvested and deciliated in 300 ml of proteose peptone with 300 mg of dibucaine. In some experiments, cells were washed three times in HNMK (50 mM Hepes, pH 6.9, 36 mM NaCI, 1 mM KCI, © The Rockefeller University Press, 0021-9525/88/12/2679/10 $2.00 The Journal of Cell Biology, Volume 107, (No. 6, Pt. 2) Dec. 1988 2679-2688 2679 on M ay 5, 2017 D ow nladed fom Published December 1, 1988