Assembly properties of tubulin after carboxyl group modification.
By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.
Mejillano
M R
MR
Department of Biochemistry, University of Kansas, Lawrence 66045.
Himes
R H
RH
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D010450:Endopeptidases; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008744:Methylamines / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure
D046911:Macromolecular Substances; D008744:Methylamines; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D014404:Tubulin; C022010:glycine ethyl ester; C027451:methylamine; D010450:Endopeptidases; D008274:Magnesium; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium; D005998:Glycine