Cloning and characterization of a gene encoding an actin-related protein in Chlamydomonas.

The genomic sequence of an actin-related gene in Chlamydomonas reinhardtii has been determined. The deduced amino acid sequence of this gene shares a 63.9% identity with that of a recently reported conventional actin-encoding gene in C. reinhardtii. Phylogenetic analysis shows that the product of this actin-related gene does not fit into conventional actin or any major actin-related protein categories. The actin-related gene in C. reinhardtii contains seven introns in the coding region and, as described for the conventional actin gene, it contains several sequences similar to the 'tub box' sequence motif in its 5'-upstream region. Southern blot analysis of the gene shows a hybridization pattern different from that of the conventional actin gene, indicating that these genes are distinct from one another. Northern blot analysis of poly(A)+RNA shows the messages of the two genes to be very similar in size, yet the message level of the actin-related gene is significantly lower than that of the conventional actin gene.

High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin.

Caltractin is a member of the calmodulin superfamily of Ca(2+)-binding proteins that was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii. Human and mouse homologs to algal caltractin have been recently characterized. In the studies reported here, recombinant Chlamydomonas caltractin was expressed at high levels in Escherichia coli and purified to homogeneity. The use of the ompT-host BL21 proved critical for obtaining high yields of homogeneous full-length protein. Growth and purification protocols were optimized to allow reproducible and efficient production of tens of milligrams of pure protein from 1-liter cultures. Caltractin has a distinct UV spectrum which is largely dominated by the fine structure due to the 9 Phe residues. Unlike other members of the same protein family, the UV and the CD spectra do not change upon addition of Ca2+ to the apoprotein. However, the 1H NMR spectrum shows distinct changes upon Ca2+ binding, which are indicative of structural and/or dynamic changes largely reminiscent of other members of the calmodulin superfamily. Ca2+ binding measurements demonstrated the binding of four Ca2+ ions to caltractin with two higher affinity (Kd = 1.2 x 10(-6) M) and two lower affinity (Kd = 1.6 x 10(-4) M) sites. Caltractin is highly stable in both the apo- and the Ca(2+)-loaded states. The unusual stability of apocaltractin makes this protein highly suited for structural studies by multidimensional NMR aimed at understanding the structural and dynamic consequences of Ca2+ binding, and the molecular basis of Ca2+ signal transduction.

Molecular cloning and centrosomal localization of human caltractin.

Caltractin, a 20-kDa calcium-binding protein, was previously purified and cloned at the DNA level from the unicellular green alga Chlamydomonas. It is a structural component of the basal body complex, the major microtubule-organizing center in Chlamydomonas and the functional homolog of the centrosome in the animal cell. Here we report the characterization of a cDNA encoding a human caltractin that shares a high degree of amino acid identity (70%) with its algal counterpart. Caltractin was identified in both HeLa and BHK cells as a 21-kDa polypeptide specifically localized to the centrosome of interphase and mitotic cells. The high level of conservation in the amino acid sequence of caltractin from algae to humans and its association with the major microtubule-organizing center in the cell suggest that caltractin plays a fundamental role in microtubule-organizing center structure and function.

Genomic structure of Chlamydomonas caltractin. Evidence for intron insertion suggests a probable genealogy for the EF-hand superfamily of proteins.

A clone containing the gene locus for Chlamydomonas caltractin, a 20,000 Mr calcium-binding protein that is a member of the EF-hand superfamily of calcium-modulated proteins, was isolated and the structural organization of the gene was determined. The intron-exon organization was resolved by direct comparison of the genomic sequence with a caltractin cDNA. The promoter region does not contain the typical TATA or CCAAT boxes, but the sequences at the splice junctions are similar to those of other eukaryotes. The positions of the six introns in the caltractin gene do not typically define unit structures, nor do they coincide with those in genes for other members of the EF-hand superfamily. An analysis of exon sequences at the splice junctions in the genes of this multigene family was undertaken; evidence was obtained that supports the hypothesis that introns arose at proto-splice sites. A probable evolutionary history for the EF-hand superfamily based on intron insertion is offered.

Missense mutations at lysine 350 in beta 2-tubulin confer altered sensitivity to microtubule inhibitors in Chlamydomonas.

Two beta-tubulin mutants of Chlamydomonas reinhardtii, colR4 and colR15, were previously isolated in our laboratory. Each mutant expressed an acidic beta-tubulin variant as a result of an alteration in the coding sequence of one of the two beta-tubulin genes in C. reinhardtii, which in the wild type encode identical proteins. In this report, we describe the identity of the specific beta-tubulin altered in the colR mutants and the precise nature of the genetic lesions. Hybrid selection of mutant poly(A)+ RNA with cDNA probes specific for the two beta-tubulins in C. reinhardtii indicated that both mutations resided in the beta 2-tubulin gene. cDNA libraries were constructed with mutant poly(A)+ RNA, and beta 2-tubulin cDNA clones were isolated. Results of in vitro transcription of cloned beta 2-tubulin cDNAs confirmed the identity of the altered genes. Sequencing of the entire coding regions of the beta 2-tubulin cDNA clones revealed that the mutants carried different single-base substitutions in the same codon for the amino acid at position 350 in the beta 2-tubulin sequence, effecting a change from a lysine to a glutamic acid in the colR4 variant and to a methionine in the colR15 variant. These changes in amino acids are consistent with the difference in the charge of the two variant polypeptides observed in isoelectric focusing. Because both the colR4 and colR15 mutations confer an altered sensitivity to a number of different microtubule inhibitors and herbicides, lysine 350 appears to be of functional importance in the structure of the tubulin molecule.

Purification and characterization of a basal body-associated Ca2+-binding protein.

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.

Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product.

An extended synthetic oligonucleotide (59-mer) was used to isolate a Chlamydomonas cDNA containing the entire coding region for a novel basal body-associated 20-kD calcium-binding protein (CaBP). DNA and RNA blot analysis indicate that the 20-kD CaBP is encoded by a single copy gene from which is derived an approximately 1.1-kb-long transcript. The deduced amino acid sequence for the protein shows a linear relatedness with calmodulin from Chlamydomonas and other organisms (45-48% identity). The primary protein sequence of the 20-kD CaBP and its predicted secondary structure suggests that the protein is likely to contain four homologous calcium-binding domains that conform to the helix-loop-helix (or EF hand) structure found in calmodulin and related calcium-modulated proteins. The major difference between the protein and calmodulin is an amino-terminal domain of 21 amino acids present on the 20-kD CaBP. In addition to its relatedness to calmodulin, the Chlamydomonas 20-kD CaBP shows a strong sequence identity (50%) with the yeast Saccharomyces cerevisiae CDC31 gene product required for spindle pole body duplication. The association of these sequence-related calcium-binding proteins to microtubule-organizing centers of divergent structure suggests a potential conserved function for the proteins.

Beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii.

Two beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii have been isolated on the basis of altered sensitivity to the growth-inhibitory effect of colchicine. The two mutations of colR4 and colR15 have been found to be tightly linked, mapping to a previously unmarked site in linkage group XII. The drug-resistance phenotypes of both mutations segregated in genetic crosses with the presence of distinct, acidic variant beta-tubulin isoforms found assembled into the microtubules of the flagella. Analysis of the in vitro translation products of total poly(A)+ RNA from the mutants provided evidence that the variant proteins are altered primary beta-tubulin gene products. Compared to wild type, strains carrying the mutations expressed an increased resistance to the inhibitory effects of colchicine in clonal growth, flagellar assembly, and germination of meiotic products, suggesting that the beta-tubulin altered in the mutants participates in multiple microtubule functions.

Possible treadmilling in zinc(II)-induced sheet polymers of bovine brain microtubule protein.

We have examined the ability of zinc(II)-induced sheet polymers, formed from thrice-cycled bovine brain microtubule protein prepared in the absence of glycerol, to exchange with tubulin subunits at steady state. By a rapid filtration assay in which labeled GTP was used as a marker for tubulin addition and loss, we found that steady-state sheet polymers, formed in 0.5 mM-ZnCl2, 1 mM-dithiothreitol, and 100 mM-2-(N-morpholino)ethanesulfonic acid (pH 6.75) in the presence of a GTP-regenerating system at 37 degrees C, incorporated the label in a time-dependent manner to a maximum level. The steady-state uptake of label was inhibited by colchicine, podophyllotoxin and vinblastine. In pulse-chase experiments, we observed that label added onto sheet polymers in a short pulse was retained for a period equal to that required by the polymers to become fully labeled in a continuous pulse; thereafter, the label was lost gradually to a baseline level. An average of 82% of the label was retained in the sheet polymers after a "cold" chase of equal duration to the time of the pulse. Sheet polymers assembled from microtubule protein prepared in the presence of glycerol gave similar results. Using a double-labeling procedure to analyze tubulin addition and loss simultaneously, we found that the rates of steady-state addition and loss were similar. Sheet polymers retained their structural integrity throughout these experiments, as determined by electron microscopy. We believe that the data are consistent with a "treadmilling" mechanism of polymerization and depolymerization, analogous to that documented to occur in steady-state microtubules in vitro. Such a mechanism is discussed in the context of recent findings from structural studies, and a model consistent with established structural data is offered.