Tubulin in sea urchin embryonic cilia: characterization of the membrane-periaxonemal matrix.
When the membranes of isolated embryonic cilia from three species of sea urchin are detergent-solubilized, the major proteins found are two equi-molar polypeptides comigrating with tubulin subunits. Cross-reactivity with a variety of tubulin antibodies confirms their identity. Calmodulin and other calcium-binding proteins are prominent minor constituents of the extract. Removal of the solubilizing detergent by adsorption to polystyrene beads, followed by a freeze-thaw cycle, produces membrane leaflets and vesicles of uniform bouyant density. Such reconstituted membranes incorporate most of the tubulin and minor proteins but not calmodulin. Equivalent cross-reactivity with antibodies to acetylated or detyrosinated alpha-tubulin indicates that the tubulin derived from the membrane-periaxonemal matrix and axoneme are indistinguishable from each other in terms of these posttranslational modifications but are distinct from the mainly unmodified tubulin of the embryonic cytoplasm. Pulse labeling with [3H]palmitate does not label either tubulin subunit but acylation does occur on a 190 x 10(3) Mr membrane protein. Its specific activity is essentially the same whether label is applied to embryos with existing or regenerating cilia, suggesting rapid ciliary membrane protein exchange or physical turnover. Using pulse-chase labeling with [3H]leucine during steady-state ciliary growth or induced regeneration in both normal and zinc-animalized embryos, the specific activity of the membrane-periaxonemal matrix-derived tubulin is initially higher than that of the axoneme but the degree of labeling equalizes in successive regenerations, consistent with derivation from a common pool. Many heavily labeled axonemal architectural proteins, such as tektin-A, are not reflected in the membrane-periaxonemal matrix fraction, suggesting that this fraction is not simply a pool of unassembled axonemal precursors.
Stephens
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Marine Biological Laboratory, Woods Hole, MA 02543.
D000818:Animals; D000906:Antibodies; D002923:Cilia / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents; D004625:Embryo, Nonmammalian / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012617:Sea Urchins / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D000906:Antibodies; D003902:Detergents; D008565:Membrane Proteins; D014404:Tubulin; D015032:Zinc