Carlos A. Arce

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE‐SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE‐INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G‐25‐40 was able, after addition of some factors, to incorporate [I4C]arginine (apparent Km= 5 μM) and [14C]tyrosine (apparent Km= 20 μM) into its own proteins. The factors required for the incorporation of [14C]arginine were: ATP (optimal concentration = 0‐25‐2 μM) and Mg2+ (optimal concentration 5 mM). For the incorporation of [I4C]tyrosine the required factors were: ATP (apparent Km= 0‐75 μM), Mg2+ (optimalconcentration 8‐16 mM) and K+ (apparent Km= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [14C]arginine and [14C]tyrosine, respectively, 7‐4 and 7‐0 in phosphate buffer and 7–9 and 7‐3‐8‐1 in tris‐HCl buffer. Pancreatic ribonuclease abolished the incorporation of [14C]arginine but had practically no effect in the incorporation of [14C]tyrosine. Furthermore, [14C]arginyl‐tRNA was a more effective donor of arginyl groups than [14C]arginine, whereas [14C]tyrosyl‐tRNA was considerably less effective than [14C]tyrosine. The incorporations of [14C]arginine and [14C]tyrosine into brain proteins were from 25‐ to 2000‐fold higher than for any other amino acid tested (12 in total). In brain [14C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [14C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [14C]Arginine was bound so that its a‐amino group remained free. Maximal incorporation of [14C]tyrosine in brain of 30‐day‐old rats was about one‐third of that in the 5‐day‐old rat. The changes with postnatal age in the incorporation of [14C]arginine were not statistically significant.

Some common properties of the protein that incorporates tyrosine as a single unit and the microtubule proteins.

Abstract Properties so far studied of the protein that incorporates tyrosine show remarkable similarities with those of the microtubule proteins. The molecular weight of proteinyl-14C-tyrosine determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 54,000. The acceptor protein and proteinyl-14C-tyrosine were found in different states of aggregation; one of these states is apparently a dimer of molecular weight approximately 110,000. From a single preparation of proteinyl-14C-tyrosine variable proportions of dimer and higher molecular weight aggregates were obtained by incubating in different conditions. Proteinyl-14C-tyrosine was eluted from DEAE-Sephadex A-50 similarly to 3H-colchicine-tubulin complex. The pattern of elution from Sephadex G-200 of dimer proteinyl-14C-tyrosine was similar to that of 3H-colchicine-tubulin complex. Proteinyl-14C-tyrosine was precipitated with vinblastine sulfate.

Posttranslational tyrosination/detyrosination of tubulin

Tubulin can be posttranslationally modified at the carboxyl terminus of the α-subunit by the addition or release of a tyrosine residue. These reactions involve two enzymes, tubulin: tyrosine ligase and tubulin carboxypeptidase. The tyrosine incorporation reaction has been described mainly in nervous tissue but it has also been found in a great variety of tissues and different species. Molecular aspects of the reactions catalyzed by these enzymes are at present well known, especially the reaction carried out by the ligase. Several lines of evidence indicate that assembled tubulin is the preferred substrate of the carboxypeptidase, whereas nonassembled tubulin is preferred by the ligase. Apparently this posttranslational modification does not affect the capacity of tubulin to form microtubules but it generates microtubules with different degrees of tyrosination. Variation in the content of the carboxyterminal tyrosine of α-tubulin as well as changes in the activity of the ligase and the carboxypeptidase are manifested during development. Changes in the cellular microtubular network modify the turnover of the carboxyterminal tyrosine of α-tubulin. Different subsets of microtubules with different degrees of tyrosination have been detected in interphase cells and during the mitotic cycle. Data from biochemical, immunological, and genetic studies have been compiled in this review; these are presented, with pertinent comments, with the hope of facilitating the comprehension of this particular aspect of the microtubule field.

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [14C]tyrosine into the C‐terminal position of α‐tubulin of rat brain cytosol was 10‐fold higher for non‐assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non‐assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot‐TCA‐insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase‐insensitive and required ATP (apparent Km= 0.64 mm), KC1 (apparent Km= 14 mm) and MgCl2 (optimal concentration range 4‐15 mm). The apparent Km for phenylalanine was 2.9 mm. [14C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [14C]phenylalanine and [14C]tyrosine when incubated simultaneously or successively. The Km and Kt (3.3 mm) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [14C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [14C]Phenylalanine and [14C]tyrosine entered into COOH‐terminal positions in the reactions described. Brain exhibited a 25‐ to 100‐fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin☆

In vitro incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [14C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [14C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [14C]tyrosine. The α and β subunits of tubulin from animals that received [14C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of αβ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [14C]amino acids was injected, the radioactivity ratio of αβ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.

Tubulin must be acetylated in order to form a complex with membrane Na + ,K + -ATPase and to inhibit its enzyme activity

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na+,K+-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na+,K+-ATPase and consequent inhibition of enzyme activity.

Activation of the plasma membrane H+‐ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H+‐ATPase–acetylated tubulin complex

In the yeast Saccharomyces cerevisiae, plasma membrane H+‐ATPase is activated by d‐glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H+‐ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H+‐ATPase–tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H+‐ATPase activity increased as a function of glucose concentration, as measured by both ATP‐hydrolyzing capacity and H+‐pumping activity. The addition of 2‐deoxy‐d‐glucose inhibited the above glucose‐induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti‐(acetylated tubulin) and anti‐(H+‐ATPase) immunoglobulins indicated a physical interaction between H+‐ATPase and acetylated tubulin in the membranes of glucose‐starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H+‐ATPase and acetylated tubulin partially colocalize at the periphery of glucose‐starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H+‐ATPase–tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H+‐ATPase activity by acetylated tubulin and concomitant increase of the H+‐ATP ase–tubulin complex.

Submembraneous microtubule cytoskeleton: regulation of ATPases by interaction with acetylated tubulin

The ATP‐hydrolysing enzymes (Na+,K+)‐, H+‐ and Ca2+‐ATPase are integral membrane proteins that play important roles in the exchange of ions and nutrients between the exterior and interior of cells, and are involved in signal transduction pathways. Activity of these ATPases is regulated by several specific effectors. Here, we review the regulation of these P‐type ATPases by a common effector, acetylated tubulin, which interacts with them and inhibits their enzyme activity. The presence of an acetyl group on Lys40 of α‐tubulin is a requirement for the interaction. Stimulation of enzyme activity by different effectors involves the dissociation of tubulin/ATPase complexes. In cultured cells, acetylated tubulin associated with ATPase appears to be a constituent of microtubules. Stabilization of microtubules by taxol blocks association/dissociation of the complex. Membrane ATPases may function as anchorage sites for microtubules.

Tentative identification of the amino acid that binds tyrosine as a single unit into a soluble brain protein

A protein system that upon addition of ATP, Mg2+ and K’ can incorporate tyrosine or phenylalanine as single units has been found in the soluble fraction of rat brain homogenate [ 1,2] . Some properties of the protein that incorporates these amino acids are similar to those generally considered distinctive of the microtubule proteins [3]. It was also reported that tyrosine &phenylalanine is bound to the carboxyl end of the protein [2], but the binding amino acid of the acceptor protein was not identified. In the work reported herein, radioactive peptides were isolated after partial acid hydrolysis of the labelled product containing [14C] tyrosine (proteinyl-[‘4C] tyrosine), in attempts to identify the dipeptide containing the labelled amino acid.