The Journal of Cell Biology, Vol 84, 717-738, Copyright © 1980 by The Rockefeller University Press

ARTICLES

Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes

A Adoutte, R Ramanathan, RM Lewis, RR Dute, KY Ling, C Kung and DL Nelson

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

• Stephan, A. B., Shum, E. Y., Hirsh, S., Cygnar, K. D., Reisert, J., Zhao, H. (2009). ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification. Proc. Natl. Acad. Sci. USA 106: 11776-11781 [Abstract] [Full Text]  
• Kim, K., Son, M., Peterson, J. B., Nelson, D. L. (2002). Ca2+-binding proteins of cilia and infraciliary lattice of Paramecium tetraurelia: their phosphorylation by purified endogenous Ca2+-dependent protein kinases. J. Cell Sci. 115: 1973-1984 [Abstract] [Full Text]  
• Redeker, V, Levilliers, N, Schmitter, J., Le Caer, J., Rossier, J, Adoutte, A, Bre, M. (1994). Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules. Science 266: 1688-1691 [Abstract]  
• Walczak, C., Nelson, D. (1993). In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium. J. Cell Sci. 106: 1369-1376 [Abstract]  
• Shih, S. J., Nelson, D. L. (1992). Proteolytic processing of secretory proteins in Paramecium: immunological and biochemical characterization of the precursors of trichocyst matrix proteins. J. Cell Sci. 103: 349-361 [Abstract]  
• Ehrlich, B., Finkelstein, A, Forte, M, Kung, C (1984). Voltage-dependent calcium channels from Paramecium cilia incorporated into planar lipid bilayers. Science 225: 427-428 [Abstract]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents