Distinct Localization and Cell Cycle Dependence of COOH Terminally Tyrosinolated a-Tubulin in the Microtubules of Trypanosoma brucei brucei

0t-Tubulin can be posttranslationally modified in that its COOH-terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of tl-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both a-tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated a-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated a-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated ~t-tubulin is a marker for newly formed microtubules. M ICROTUBULES (MTs) 1 can exert a number of different functions, assume various configurations in different cellular compartments, and exhibit vastly different stabilities even within individual cells. A number of biochemical events are thought to contribute to the amazing structural and functional flexibility of these rigidly conserved polymers. These include the variation in the activity and localization of microtubule-organizing centers (Tucker, 1979), changing properties and local concentrations of microtubule-associated proteins, and the presence within different MTs of tubulins with differing amino acid sequences (Ponstingl et al., 1982; Cleveland and Sullivan, 1985). A further means of generating MT heterogeneity is via posttranslational modification of the tubulin proteins themselves (Eipper, 1974; Sandoval and Cuatrecasas, 1976; Feit and Shelansky, 1975; UHernault and Rosenbaum, 1983). An unusual type of modification has been observed by Barra et al. (1973), who reported the posttranslational addition of tyrosine to a brain protein which was subsequently identified as ct-tubulin. Further studies have demonstrated that the tyrosine is added to the carboxy terminus of ct-tubulin. In most organisms, ct-tubulin is initially synthesized with a tyrosine as its COOH-terminal amino acid (Tyrtubulin) (Valenzuela et al., 1981; Cleveland and Sullivan, 1985). This tyrosine can be posttranslationally removed in vivo by a specific carboxy-peptidase (Argarana et al., 1980; 1. Abbreviation used in this paper: MT, microtubule. Martensen, 1982), exposing the penultimate amino acid residue, which is glutamic acid (Glu-tubulin). Most likely, the substrate for this enzyme activity is the tubulin of the MTs (Thompson et al., 1979; Kumar and Flavin, 1981). Such detyrosinolated a-tubulin then becomes the substrate for a cytoplasmic tubulin tyrosine ligase which restores a tyrosine residue at the COOH terminus of et-tubulin (Tyr-tubulin) (Raybin and Flavin, 1975; Raybin and Flavin, 1977; Thompson, 1982; Flavin and Murofushi, 1984). This enzyme has recently been purified to homogeneity (Schr6der et al., 1985). Tubulin tyrosine ligase activity has been demonstrated in cell lysates from vertebrates (Preston et al., 1979) as well as from a number of invertebrates (Kobayashi and Flavin, 1981; Gabius et al., 1983), but no such activity has yet been detected in unicellular organisms. Nevertheless, the in vivo occurrence of posttranslational tyrosinolation of ct-tubulin has recently been demonstrated for the hemoflagellate Trypanosoma brucei (Stieger et al., 1984). Remarkably, in this organism both the ctand the 13-tubulins carry a tyrosine as their COOH-terminal amino acid (Kimmel et al., 1985), but only the a-tubulin can serve as a substrate for the posttranslational tyrosinolation reaction. Despite much effort, very little is understood about the functional significance of the detyrosinolation/tyrosinolation reaction cycle. Changes in COOH-terminal tyrosine content or in tubulin tyrosine ligase activity have been correlated with changes in cell shape (Deanin et al., 1981), cell differen© The Rockefeller University Press, 0021-9525/87/03/439/8 $1.00 The Journal of Cell Biology, Volume 104, March 1987 439--446 439 on July 6, 2017 jcb.rress.org D ow nladed fom tiation (Nath and Flavin, 1979; Cumming et al., 1984), redox status of the cell (Nath and Gallin, 1984), progression through the cell cycle (Forrest and Klevecz, 1978), and ageing (Gabius et al., 1983). Also, a role for tyrosinolated ct-tubulin in the functioning of voltage-gated sodium channels has been proposed (Matsumoto et al., 1984a, b). Immunocytochemical studies using monoclonal antibodies specific for the COOH-terminal tyrosine (Kilmartin et al., 1982) or polyclonal antibodies raised against synthetic peptides representing the tyrosinolated as well as the detyrosinolated COOH terminus of ct-tubulin (Gundersen et al., 1984) have led to a general picture in which most microtubules are seen to contain both tyrosinolated and detyrosinolated a-tubulin, though in varying ratios (Wehland et al., 1983; Gundersen et al., 1984). In some instances, individual microtubules have been detected which seem to contain exclusively one or the other of the two forms of ct-tubulin (Cumming et al., 1984; Gundersen et al., 1984). Trypanosomes are attractive for investigating a possible correlation between the tyrosinolation status of ct-tubulin and microtubular localization and/or function. Interphase cells of these organisms contain three distinct populations of MTs; namely (a) the cytoplasmic tubulin pool, (b) the subpellicular array of membrane-associated singlet MTs, and (c) the MTs of the flagellar axoneme. These microtubular structures can be visualized by electron microscopy (Angelopoulos, 1970) and can be biochemically separated with relative ease (Russell et al., 1984; Russell and Gull, 1984; Schneider et al., 1987). The present report demonstrates that tyrosinolated ~t-tubulin is present in all three cellular compartments outlined above, and that both ct-tubulin isoforms present in trypanosomes (Schneider et al., 1987) can be posttranslationally tyrosinolated. Immunofluorescence studies show that tyrosinolated ct-tubulin is nonrandomly distributed in the MTs. Our observations suggest a temporal control of tubulin tyrosinolation through the cell cycle. Materials and Methods The procedures used in this study are described in the preceding paper (Schneider et al., 1987). Conditions for inhibiting protein synthesis, for in vivo labeling of trypanosomes with [3H]tyrosine, and for Taxol-induced polymerization of tubulin were exactly as described earlier (Stieger et al.,