Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate

Abstract

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.

The Majority of the Saccharomyces cerevisiae Septin Complexes Do Not Exchange Guanine Nucleotides

“…To identify which polypeptides are responsible for the observed in vitro GTP-binding activity, we incubated our septin preparation with [␣-32 P]GTP for different amounts of time (0 and 5 h) and then UV cross-linked the septin complex to the bound radioactive nucleotide (27). The identity of the crosslinked proteins was determined by SDS-PAGE and phosphorimager analysis (Fig.…”

“…These data strongly support a structural role for septin-bound GTP and GDP, analogous to the role of GTP for ␣-tubulin. In the case of ␣-tubulin, GTP is non-exchangeable (27,28) and thought to be important for ␣-tubulin folding and tubulin dimer assembly. Similarly, septin-bound GTP and GDP might be important for individual septin protein folding and/or septin complex assembly.…”

“…GTP might turn over rapidly and regulate the function of septins, as in the cases of ␤-tubulin (24,25), FtsZ (26), and small GTPases. Alternatively, GTP might incorporate once during folding or complex assembly and play no further role in the function of septins, analogous to GTP bound to ␣-tubulin (27,28). In vitro experiments implicate GTP binding in filament formation by an individual Xenopus septin (22) and possibly in the interaction between different septin polypeptides (23).…”

Photoaffinity labeling of the nucleotide‐binding site of the uncoupling protein from hamster brown adipose tissue

“…It should be noted that the sensitivity of UCP to far dtraviolet irradiation also aborted experiments to covaleni ly incorporate unsubstituted GTP or ATP into UCP by strong ultraviolet irradiation. This procedure has been applied, for example, in labeling tubulin with GTP [14].…”

A model of the nucleotide‐binding site in tubulin

“…Tubulin binds two molecules of GTP tightly: one exchangeably, at the so-called E-site in fltubulin [8,9], and one non-exchangeably, presumably at a site in cr-tubulin. One Mg 2+ is also bound tightly, either indirectly as a nucleotidemetal ion complex [10] or possibly directly to certain residues in the protein [11].…”