Use of a heat-stable microtubule-associated protein class-specific antibody to investigate the mechanism of microtubule binding.
Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule-binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat-stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val-Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co-sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding.
Dingus
J
J
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Obar
R A
RA
Hyams
J S
JS
Goedert
M
M
Vallee
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D003429:Cross Reactions; D000939:Epitopes; D006358:Hot Temperature; D015151:Immunoblotting; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008869:Microtubule-Associated Proteins