Polyglutamylation of Atlantic cod tubulin: Immunochemical localization andpossible role in pigment granule transport

In higher organisms, there is a large variety of tubulin isoforms, due to m ultiple tubulin genes and extensive post-translational modification. The pr operties of microtubules may be modulated by their tubulin isoform composit ion. Polyglutamylation is a post-translational modification that is thought to influence binding of both structural microtubule associated proteins (M APs) and mechano-chemical motors to tubulin. The present study investigates the role of tubulin polyglutamylation in a vesicle transporting system, co d (Gadus morhua) melanophores. We did this by microinjecting an antibody ag ainst polyglutamylated tubulin into these cells. To put our results into pe rspective, and to be able to judge their universal application, we characte rized cod tubulin polyglutamylation by Western blotting technique, and comp ared it to what is known from mammals. We found high levels of polyglutamyl ation in tissues and cell types whose functions are highly dependent on int eractions between microtubules and motor proteins. Microinjection of the an ti-polyglutamylation antibody GT335 into cultured melanophores interfered w ith pigment granule dispersion, while dynein-dependent aggregation was unaf fected. Additional experiments showed that GT335-injected cells were able t o aggregate pigment even when actin filaments were depolymerized, indicatin g that the maintained ability of pigment aggregation in these cells was ind eed microtubule-based and did not depend upon actin filaments. The results indicate that dynein and the kinesin-like dispersing motor protein in cod m elanophores bind to tubulin on slightly different sites, and perhaps depend differentially on polyglutamylation for their interaction with microtubule s. The binding site of the dispersing motor may bind directly to the polygl utamate chain, or more closely than dynein. Cell Motil. Cytoskeleton 44:263 -273, 1999. (C) 1999 Wiley-Liss, Inc.