Tubulin amino acid sequences are highly conserved across diverse eukaryotes such as budding yeast, nematodes, Arabidopsis and humans. Constraints on sequence variation arise from the requirement that individual tubulins maintain interactions with GTP as well as with a multitude of proteins including other tubulins, motors and microtubule associated proteins. The observation that the same or similar substitutions to tubulin have arisen in dramatically different circumstances and organisms, implies that a systematic analysis of tubulin point mutations may help explain how individual amino acids and domains in tubulins contribute to overall protein function in these essential subcellular machines.
Tubulins are a group of conserved globular proteins which carry out essential functions in eukaryotes. The tubulin superfamily contains six families of related proteins that arose from a single ancestral protein. All tubulins have a characteristic GTP binding domain.
Each tubulin family has a specialized role, with the most known about the function of α‑ and β‑tubulin which polymerize to form microtubules. The structure of α‑β‑tubulin dimers and the organization of dimers in microtubules is known. Microtubule assembly is initiated by addition of α‑β‑tubulin dimers onto a scaffold formed by the γ‑tubulin ring complex (γ‑TuRC). Many eukaryotes have more than one gene encoding slightly different isotypes of α‑, β‑ and γ‑tubulins. For example, humans have eight α‑tubulin genes, nine β‑tubulin genes, and two γ‑tubulin genes. Isotypes are not completely interchangeable and varied expression levels of individual α‑ and β‑tubulin isotypes permits specific cell types to fine‑tune microtubule properties. While α‑, β‑ and γ‑tubulins are essential in all eukaryotes, δ‑, ε‑ and ζ‑tubulins are only found in eukaryotes that construct appendage‑containing centrioles.
The α‑tubulin subunit associates with β‑tubulin to form a stable heterodimer which is the subunit that polymerizes to form microtubules. Heterodimers stack head to tail to form protofilaments and thirteen of these associate laterally to form most native microtubules. The heterodimer orientation creates polarity in the microtubule: α‑tubulin is exposed at the slow growing “minus” end which is typically embedded in a microtubule organizing center (MTOC). Since α‑ and β‑tubulin are related, they share sequence similarity and overall structural similarity. There are also important differences in their structure and function. Both α‑ and β‑tubulin bind to GTP, but α‑tubulin‑bound GTP is non‑exchangeable as the opening to the binding site is blocked by the monomer‑monomer interface with β‑tubulin. The α‑tubulin subunit has a GTPase activating (GAP) domain that acts on β‑tubulin in the adjacent dimer within an individual microtubule protofilament such that GTP is hydrolyzed to GDP in a polymerization‑dependent fashion. Subunit interactions in the microtubule are stabilized by an eight amino acid insert in α‑tubulin that is absent from β‑tubulins. Few of the large number of tubulin binding drugs specifically associate with α‑tubulin. Only pironetin has been confirmed to bind to α‑tubulin, although dinitroanilines such as oryzalin are also predicted to bind to this subunit.
The β‑tubulin subunit associates with α‑tubulin to form a stable heterodimer which polymerizes with β‑tubulin exposed at the rapidly growing “plus” end of microtubules. β‑tubulin bound GTP is hydrolyzed to GDP in a polymerization‑dependent fashion: a GAP domain in α‑tubulin of the adjacent dimer induces β‑tubulin to hydrolyze GTP. The conformation of GTP‑bound heterodimers favors microtubule assembly, while the conformation of dimers that contain β‑tubulin bound to GDP favors disassembly. Most characterized drugs bind to β‑tubulin: these include microtubule stabilizing drugs such as paclitaxel and destabilizing drugs such as colchicine, vinblastine and the cryptophycins.
The γ‑tubulin subunit is a component of diverse microtubule organizing centers. Mutational analysis in a variety of organism suggests that GTP hydrolysis is critical for γ‑tubulin function. Directed mutations to γ‑tubulin have probed the role of conserved residues that likely coordinate GTP binding and hydrolysis while human γ‑tubulin mutations are associated with defects in neural development.
Genes for δ‑, ε‑ and ζ‑tubulins are found in organisms that construct appendage‑containing centrioles and are missing from organisms that lack centrioles (higher land plants and most fungi) and organisms with atypical centrioles that lack appendages (such as C. elegans and D. melanogaster).
Tubulin mutations have been identified by a number of methods. The earliest mutations to tubulin were identified by screening for temperature‑sensitive growth or resistance to microtubule disrupting or stabilizing drugs. Tubulin mutations were subsequently identified in a wide variety of phenotypic screens such as screening for loss of touch receptor function in C. elegans or shoot, leaf and flower spiraling in A. thaliana. The α‑, β‑ and γ‑ tubulin genes have been analyzed by directed mutations to assess the contribution of specific amino acids to tubulin function. Tubulin mutations arise as compensatory mutations that reduce the deleterious effects of other mutations, both in tubulin and in other loci. In the past few years, it has become clear that spontaneous mutations to tubulin underlie a variety of human genetic disorders, collectively referred to as tubulinopathies. Remarkably, the same mutations appear in distinct species, tubulin isotypes, and selection methods. By identifying intersecting tubulin mutations, we can better understand the role of specific amino acids in conserved tubulin functions.
Three tables exhaustively describe characterized tubulin mutations. For each tubulin type, the point mutations are listed in order of amino acid position in the specific tubulin. Individual entries include hyperlinks to NCBI or Uniprot sequences and to specific primary literature references. Key information for each point mutation (approximate position in existing tubulin structures, species, and mutant phenotype) is displayed on a single line in the table. Other information (the substitution represented in three letter amino acid code and associated references) is displayed when an individual entry is opened. The substitution is represented in single letter amino acid code with the wild type and mutant residues color‑coded to represent their chemical properties: small nonpolar amino acids (glycine, alanine, serine, threonine) are orange; hydrophobic amino acids (cysteine, valine, isoleucine, leucine, proline, phenylalanine, tyrosine, methionine, tryptophan) are green; polar amino acids (asparagine, glutamine, histidine) are purple; negatively charged amino acids (aspartic acid, glutamic acid) are red and positively charged amino acids (lysine, arginine) are blue. Species within the major eukaryotic lineages are also color‑coded: metazoans are listed in red, higher land plants are listed in green, protozoans are listed in blue, fungi are listed in orange. The database is key word searchable, individual subsets can be displayed as sorted for organism or tubulin gene. In addition, the entire dataset or selected mutations of interest can be downloaded as pdf files.
To date, we have compiled information on 489 mutations in α‑tubulin, 736 mutations in β‑tubulin and 343 mutations in δ‑, ε‑ and ζ‑tubulins. Since we are most interested in how substitutions to specific residues alter tubulin function, we have not included data on gene silencing, targeted gene deletions or alterations in isotype expression levels in the absence of point mutations.