Production and utilization of detyrosinated tubulin in developingArtemialarvae: evidence for a tubulin-reactive carboxypeptidase

Abstract

The reversible, enzymatically driven removal and readdition of its carboxy-terminal tyrosine are major posttranslational modifications of α-tubulin. To study these processes isoform-specific antibodies were produced and subsequently used to characterize tyrosinated and detyrosinated tubulin in the brine shrimp, Artemia. Tyrosinated tubulin existed in relatively constant amounts on western blots of cell-free protein extracts from Artemia at all developmental stages examined, whereas detyrosinated tubulin was present after 20–24 h of postgastrula growth. In agreement with the blots, the detyrosinated isoform was observed in immunofluorescently stained larvae after 24 h of incubation, appearing first in structures of a transient nature, namely spindles and midbodies. The elongated muscle cells encircling the gut and the epithelium bordering the gut lumen were stained extensively with antibody to detyrosinated tubulin. Detyrosination was accompanied by the appearance of a tubulin-reactive carboxypeptidase, which used both nonpolymerized and polymerized tubulin as substrate. The enzyme bound to microtubules very poorly, if at all, under conditions used in this work. Several inhibitors of carboxypeptidase A had no effect on the carboxypeptidase from Artemia and revealed similarities between this enzyme and others thought to be tubulin specific. The use of inhibitors also indicated that the carboxypeptidase from Artemia recognized aspects of tubulin structure in addition to the carboxyterminal tyrosine. Our results support the idea that detyrosinated tubulin appears in microtubules of varying stability, and they demonstrate that Artemia possess a carboxypeptidase with the potential to detyrosinate tubulin during growth of larvae.Key words: tubulin, detyrosination, carboxypeptidase, Artemia.