Abstract.A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain mbulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubnlin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer. ETYLATIOIN of several proteins is known to occur on the epsilon-amino group of their lysine residues (I, 2, 4). Although reversible acetylation has been studied most intensively with histories, in vivo and in vitro, the regulation of this modification and its physiological role still remain to be clarified. Recently we showed, by labeling in vivo with 3H-acetate, that the acetylation and deacetylation of alpha-tubulin occur in Chlamydomonas during flagellar regeneration and resorption, respectively (3, 7-10). Acetylated alpha-tubulin was found almost exclusively in the flagellar apparatus, whereas cytoplasmic alpha-tubulin was found predominantly in the nonacetylated form (3,8,12).These results from labeling in vivo suggested that Chlamydomonas contains an alpha-tubulin acetyltransferase (TAT) 1 and a tubulin deacetylase (TDA) which carry out the reversible acetylation. Moreover, the results also indicated that there must be a mechanism by which acetylated alpha-tubulin accumulates principally in the flagellum.Recently, we have demonstrated the presence of a TAT activity, using brain tubulin as a substrate, in flagella isolated from Chlamydomonas (5). We have purified and characterized the flagellar TAT to understand the role of alpha-tubulin acetylation in flagellar formation and/or function, and to determine the mechanism by which the acetylated alphatubulin is accumulated specifically in the flagellum. In this paper we show that (a) the TAT is highly specific for alpha-1. Abbreviation~ uted in this paper: DTT, dithiotb_reitoI; MAPs, mAcrotubule-associated proteins; MTP, microtubule proteins; PC, phosphoeeltnlose; TAI, tubulin acelylation inhibitor; TAT, alpha-tubulin acetyhransferase; TDA, tubulin deacetylase. tubulin, is tightly bound to the flageilar axoneme, and can be released from the axoneme...

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A number of studies suggest that several members of the transforming growth factor‐β (TGF‐β) family of peptide growth factors may be involved in the regulation of cartilage differentiation. It has been previously reported that TGF‐β1 and TGF‐β2 promote the chondrogenic differentiation of chick limb mesenchymal cells in high density micromass cultures (Kulyk et al. [1989a] Dev. Biol. 135:424–430). In this study we report that chick limb mesenchymal cells express mRNA for chicken TGF‐β1, TGF‐β2, and TGF‐β3 during cartilage differentiation in vitro. In addition, the time course of their expression during cartilage differentiation is consistent with their playing a role in the initiation of this differentiation process. We also report that two members of the TGF‐β family, TGF‐β3 and bone morphogenetic protein‐2 (BMP‐2), are capable of promoting the accumulation of cartilage extracellular matrix molecules by differentiating chick limb mesenchymal cells in micromass culture. Significant differences, however, were noted between the specific effects on matrix production elicited by these two growth factors which suggest that they may be acting by distinct mechanisms to regulate cartilage matrix production. TGF‐β appears to be most effective on cells which have not yet undergone cell condensation, a critical event in early cartilage differentiation, wherease BMP‐2 is most effective after cells have condensed or differentiated. These observations suggest that TGF‐β3 and BMP‐2 may be acting in a sequential manner to regulate chick limb mesenchymal cells through the different stages of cartilage differentiation. © 1994 Wiley‐Liss, Inc.

We have previously shown that the a-tubulin of Chlamydomonas flagella is synthesized as a precursor which is modified by acetylation in the flagellum during flagellar assembly. In this report, we show the presence of an t~-tubulin acetylase activity in isolated Chlamydomonas flagella that is highly specific for o~-tubulin of both mammalian brain and Chlamydomonas.Detachment of the flagella of the bi-flagellate alga Chlamydomonas stimulates the synthesis and accumulation of mRNAs for tubulin and other flagellar proteins (1--4). Translation of these mRNAs in a reticulocyte lysate system showed the flagellar a-tubulin to be made as a precursor (a~), slightly more basic than the mature a-tubulin (a3) found in the assembled microtubules of flagella (5-7). The a-tubulin modification was identified as an acetylation by inhibiting protein synthesis while allowing the flagella to regenerate in the presence of radioactive acetate. Isolation of these flagella and twodimensional gel electrophoretic analysis indicated that, of more than 150 flagellar proteins, only a-tubulin became labeled (8), and that this labeling was due to an acetylation of the ~-amino group of lysine (9). If flagellar assembly was completely inhibited by colchicine, flagellar a-tubulin precursor (a~) accumulated in the cell body (10, l 1); upon release from colchicine inhibition of assembly, the a~ precursor was then found in the detergent-soluble "matrix" of the flagellum (10), presumably on its way to the tip assembly site (12-14). Thus, flagellar a-tubulin is made as a precursor which can be found in the cell body; it moves up the shaft of the flagellum as a precursor and it is changed by acetylation to the mature a3-tubulin, either just prior to or at the time of microtubule assembly at the flagellar tip. Because of these results, it was reasonable to analyze isolated flagella for a-tubulin acetylase activity. The following report shows that this enzymatic activity is present in the flagella and that it specifically acetylates a-tubulin of Chlamydomonas and mammalian brain. MATERIALS AND METHODS Preparation of Brain Microtubule Protein: Microtubule proteinwas prepared from calf brain by the method of Sloboda et al. (18) and stored as a pellet at -80"C. Before use in an acetylation assay, the microtubule protein was thawed in a 37"C water bath with 0.5 mM fresh GTP added (final GTP concentration 2.5 mM) and carried through a final cycle of temperaturedependent polymerization/depolymerization in PM buffer (100 mM PIPES, pH 6.9, with KOH, 1 mM MgSO4, 2 mM GTP, and 2 mM EGTA). For some experiments, PM buffer contained 4 M glycerol. Purified porcine tubulin was prepared by phosphocellulose chromatography (18). Assay for In Vitro Acetyfation of a-Tubulin:Acetylation of atubulin was assayed by measuring the incorporation of tritium from [3H]-acetyl-CoA into trichloroacetic acid-precipitable protein. In a typical reaction, brain microtubule protein at 2--4 mg/ml in PM buffer was mixed with an equal volume of flagella at 1-4 mg/ml in PM buffer. F...

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