Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures.

Abstract

In cultured muscle cells the carboxyl-terminal tyrosine of a-tubulin was shown to exchange rapidly with free tyrosine. The rapid turnover of this residue was dependent upon the presence of intact microtubules. Half-life determinations were made by two methods: (i) the cells were pulse-labeled in hypertonic medium, in which the major tyrosine incorporation was post-translational, and then chased with isotonic medium; and (ii) the cells were pulsed and chased in isotonic medium, and the post-translational component of the radioactivity of purified a-tubulin was calculated. Both methods yielded a half-life of 37 min or less for the terminal tyrosine residue, whereas the half-life of tubulin itself was shown to be greater than 48 hr. A rapid assembly and disassembly of several filamentous intracellular structures is coordinated with changes in cellular function and growth (1-3). The mechanisms that regulate these processes are not known, but attention has been focused on post-translational events because, at least in the case of actin and tubulin, de novo protein synthesis is not an essential aspect of this dynamic process (4,5).In this laboratory we have been studying a specific posttranslational modification of tubulin that may be involved in the control of tubulin utilization in vivo. Tubulin:tyrosine ligase (TTLase) catalyzes the addition of single units of tyrosine (or phenylalanine) to the carboxyl terminus of a-tubulin (6). TTLase activity is widely distributed in avian and mammalian tissues (7). The specific activity of the TTLase has been shown to undergo marked changes during the development of brain, liver, muscle, and sensory ganglia of the chicken, the highest activities occurring in the embryo (8, 9). The periods of high TTLase activity in brain, sensory ganglia, and muscle are temporally related to development of cellular asymmetries. Although the biochemical reaction that is catalyzed by TTLase has been characterized in vitro (10) and shown to occur in vivo (11), the significance of the modification of tubulin and the high TTLase levels to cellular function and embryonic development is not yet established. Rodriguez and Borisy (12) have recently reported that the degree of tyrosination of a-tubulin varies as a function of brain development in a manner that correlates with the changes in TTLase activity. We have speculated that the rate of turnover of the terminal tyrosine residue might be of more significance to cellular function than the absolute degree of tubulin tyrosination (8).In this paper, we examine the rate of turnover of the carboxyl-terminal tyrosine in primary cultures of chicken breast muscle, which maintain high levels of TTLase (unpublished observations). Estimates were obtained from two different types of pulse-chase experiments. The first utilized hypertonic culture medium to inhibit translational incorporation of tyrosine by blocking the formation of initiation complexes (13). Cells Npulsed with [3H]tyrosine in hypertonic medium selectively incorporate the label at the carbo...

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei.

“…Once incorporated into the polymer, Tyr-tubulin is gradually detyrosinolated by the action of the MT-associated tubulin carboxypeptidase. This is consistent with the known substrate specificity and subcellular localization of tubulin tyrosine ligase (Schrtder et al, 1985) and of tubulin carboxypeptidase (Thompson et al, 1979;Kumar and Flavin, 1981). Furthermore, it is strongly supported by recent observations (Gun-dersen et al, 1985) that in mammalian cells recovering from Nocodazole-induced loss of MTs, the et-tubulin of all newly forming MTs is heavily tyrosinolated.…”

Intracellular Signaling Molecules Activated by Epstein-Barr Virus for Induction of Interferon Regulatory Factor 7

“…As shown in Fig. 7, tubulin and EBNA-1 had very long half-lives, while IRF-1 had a very short half-life, as expected (5,43,46). Because the half-lives of both EBNA-1 and tubulin are more than 24 h (5, 43), and because the expression pattern of IRF-7 protein after cycloheximide block is similar to that of EBNA-1 and tubulin (Fig.…”

Developmental and Biochemical Analysis of Chick Brain Tubulin Heterogeneity

“…Phosphorylation of the p chain (Forgue and Dahl, 1978) and cyclic detyrosinatiodtyrosination of the a chain (e.g., see Thompson et al, 1979) have been described for neural tubulins . The tyrosination cycle of a has been ruled out, however, as a source of isoelectric heterogeneity (Gozes and Littauer, 1978).…”