All tubulins are not alike: Heterodimer dissociation differs among different biological sources

Felipe Montecinos-Franjola,Sumit K Chaturvedi,Peter Schuck,Dan L Sackett

doi:10.1074/jbc.ra119.007973

Abstract

Tubulin, the subunit of microtubules, is a noncovalent heterodimer composed of one α- and one β-tubulin monomer. Both tubulins are encoded by multiple genes or composed of different isotypes, which are differentially expressed in different tissues and in development. Tubulin αβ dimers are found throughout the eukaryotes and, although very similar, are known to differ among organisms. We seek to investigate tubulins from different tissues and different organisms for a basic physical characteristic: heterodimer stability and monomer exchange between heterodimers. We previously showed that mammalian brain tubulin heterodimers reversibly dissociate, following the mass action law. Dissociation yields native monomers that can exchange with added tubulin to form new heterodimers. Here, we compared the dissociation of tubulins from multiple sources, including mammalian (rat) brain, cultured human cells (HeLa cells), chicken brain, chicken erythrocytes, and the protozoan Leishmania We used fluorescence-detected analytical ultracentrifugation to measure tubulin dissociation over a >1000-fold range in concentration and found that tubulin heterodimers from different biological sources differ in Kd by as much as 150-fold under the same conditions. Furthermore, when fluorescent tracer tubulins from various sources were titrated with unlabeled tubulin from a single source (rat brain tubulin), heterologous dimerization occurred, exhibiting similar affinities, in some cases binding even more strongly than with autologous tubulin. These results provide additional insight into the regulation of heterodimer formation of tubulin from different biological sources, revealing that monomer exchange appears to contribute to the sorting of α- and β-tubulin monomers that associate following tubulin folding.

Highlights

Tubulin, the subunit of microtubules, is a noncovalent heterodimer composed of one ␣- and one ␤-tubulin monomer
These results provide additional insight into the regulation of heterodimer formation of tubulin from different biological sources, revealing that monomer exchange appears to contribute to the sorting of ␣- and ␤-tubulin monomers that associate following tubulin folding
Tubulin dimer dissociation detected by sedimentation velocity analytical centrifugation We wished to determine whether the properties of reversible dimer dissociation, monomer stability, and monomer exchange, which we previously demonstrated for rat brain tubulin [13], are shared with other tubulins

Summary

The abbreviations used are

FtsZ, filamentous temperature-sensitive protein; CCT, chaperonin containing TCP1; PTM, post-translational modification; RBT, rat brain tubulin; RBC, red blood cell; BtubA/B, bacterial tubulin A/B; NIS, noninteracting surface. Tubulin from brain is heterogeneous in its composition of ␣ and ␤ isotypes [20, 21] and is subject to many types of post-translational modifications that can modulate the protein’s in vitro behavior [22]. Other tubulins, isolated from different tissues or organisms, differ in isotype composition of ␣ and ␤ chains and in the content of PTMs [23], and all of these could alter dimer dissociation. We show that tubulins from five independent biological origins (cells, tissues, and organisms) all share the following properties. All tubulin dimers reversibly dissociate; the free monomers all share the same hydrodynamic properties, indicating that they retain their native fold for at least a few hours; and all can undergo monomer exchange with dimers from heterologous origins

Results

Discussion

Experimental procedures