Tubulin and tektin in sea urchin embryonic cilia: pathways of protein incorporation during turnover and regeneration

Abstract

ABSTRACT Axonemal precursor tubulin is the major protein component of the detergent-soluble membrane/matrix fraction of sea urchin embryonic cilia. Its unusual abundance may reflect the rapid turnover of these cilia, a process that is further documented here. However, whether during induced regeneration or normal turnover and growth, most other newly synthesized axonemal proteins are not detectable in the membrane/matrix fraction, raising the question of how non-tubulin precursors transit the growing cilium to the distal tip where assembly is generally thought to occur. Three potential explanations were con-sidered: (1) the assembly of these components is proximal; 2 their relative concentration is too low to detect; or (3) tubulin alone is conveyed via a membrane/matrix pathway while most other axonemal proteins are transported in association with the axoneme. Light microscope autoradi-ography of axonemes pulse-chase labeled with [3H]leucine showed relatively uniform labeling, with no evidence for proximal incorporation. Fully grown cilia and cilia at early stages of regeneration were isolated from labeled embryos, fractionated into membrane/matrix, axonemal tubulin and architectural remnant components, and their labeled protein compositions were compared. Heavily labeled axonemal proteins, most notably the integral microtubule doublet component tektin-A, were not detected in the membrane/matrix fraction of emerging cilia, even though nearly half of the total ciliary tubulin appeared in that fraction, arguing against membrane-associated or soluble matrix transit for the architectural proteins at low con-centrations. However, after thermal fractionation of axonemes from growing cilia, labeled proteins characteris-tic of the architectural remnant dominated the solubilized microtubule fraction, supporting axoneme-associated transport of the non-tubulin proteins during growth, in contrast to a membrane/matrix pathway for tubulin.