Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2⁺ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2⁺; overexpression of atb2⁺ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.     

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Free beta-tubulin not in heterodimers with alpha-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free beta-tubulin. This study focused specifically on the function of Rbl2p, which, like alpha-tubulin, can rescue cells from free beta-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of beta-tubulin relative to alpha-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of beta-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to beta-tubulin. The data suggest that Rbl2p binds transiently to free beta-tubulin, which then passes into an aggregated form that is not toxic.     

Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome

Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.     

Novel α-Glucosidase from Aspergillus nidulans with Strong Transglycosylation Activity

Aspergillus nidulans possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.     

Dominant-Lethal α-Tubulin Mutants Defective in Microtubule Depolymerization in Yeast

The dynamic instability of microtubules has long been understood to depend on the hydrolysis of GTP bound to beta-tubulin, an event stimulated by polymerization and necessary for depolymerization. Crystallographic studies of tubulin show that GTP is bound by beta-tubulin at the longitudinal dimer-dimer interface and contacts particular alpha-tubulin residues in the next dimer along the protofilament. This structural arrangement suggests that these contacts could account for assembly-stimulated GTP hydrolysis. As a test of this hypothesis, we examined, in yeast cells, the effect of mutating the alpha-tubulin residues predicted, on structural grounds, to be involved in GTPase activation. Mutation of these residues to alanine (i.e., D252A and E255A) created poisonous alpha-tubulins that caused lethality even as minor components of the alpha-tubulin pool. When the mutant alpha-tubulins were expressed from the galactose-inducible promoter of GAL1, cells rapidly acquired aberrant microtubule structures. Cytoplasmic microtubules were largely bundled, spindle assembly was inhibited, preexisting spindles failed to completely elongate, and occasional, stable microtubules were observed unattached to spindle pole bodies. Time-lapse microscopy showed that microtubule dynamics had ceased. Microtubules containing the mutant proteins did not depolymerize, even in the presence of nocodazole. These data support the view that alpha-tubulin is a GTPase-activating protein that acts, during microtubule polymerization, to stimulate GTP hydrolysis in beta-tubulin and thereby account for the dynamic instability of microtubules.     

GCP5 and GCP6: Two New Members of the Human γ-Tubulin Complex

The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.     

Formation and Function of the Rbl2p–β-Tubulin Complex

The yeast protein Rbl2p suppresses the deleterious effects of excess β-tubulin as efficiently as does α-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with β-tubulin that does not contain α-tubulin, thus defining a second pool of β-tubulin in the cell. Formation of the complex depends upon the conformation of β-tubulin. Newly synthesized β-tubulin can bind to Rbl2p before it binds to α-tubulin. Rbl2p can also bind β-tubulin from the α/β-tubulin heterodimer, apparently by competing with α-tubulin. The Rbl2p–β-tubulin complex has a half-life of ~2.5 h and is less stable than the α/β-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing β-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p–β-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.     

α-, β-, and γ-Tubulin Polymerization in Response to DNA Damage

Microtubules provide structural support for a cell and play key roles in cell motility, mitosis, and meiosis. They are also the targets of several anticancer agents, indicating their importance in maintaining cell viability. We have investigated the possibility that alterations in microtubule structure and tubulin polymerization may be part of the cellular response to DNA damage. In this report, we find that γ-radiation stimulates the production and polymerization of α-, β-, and γ- tubulin in hematopoeitic cell lines (Ramos, DP16), leading to visible changes in microtubule structures. We have found that this microtubule reorganization can be prevented by caffeine, a drug that concomitantly inhibits DNA damage-induced cell cycle arrest and apoptosis. Our results support the idea that microtubule polymerization is an important facet of the mammalian response to DNA damage.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

γ-Tubulin: The hub of cellular microtubule assemblies

In eukaryotic cells a specialized organelle called the microtubule organizing center (MTOC) is responsible for disposition of microtubules in a radial, polarized array in interphase cells and in the spindle in mitotic cells. Eukaryotic cells across different species, and different cell types within single species, have morphologically diverse MTOCs, but these share a common function of organizing microtubule arrays. MTOCs effect microtubule organization by initiating microtubule assembly and anchoring microtubules by their slowly growing minus ends, thus ensuring that the rapidly growing plus ends extend distally in each microtubule array. The goal is to define molecular components of the MTOC responsible for regulating microtubule assembly. One approach to defining the molecules responsible for MTOC function is to look for molecules common to all MTOCs. A newly discovered centrosomal protein, γ-tubulin, is found in MTOCs in cells from many different organisms, and has several properties which make it a candidate for both initiation of microtubule assembly and anchorage. The hypothesis that γ-tubulin plays a role in MTOCs in microtubule initiation and anchorage is currently being tested by a variety of experimental approaches.     

Characterization of the Human Homologue of the Yeast Spc98p and Its Association with γ-Tubulin

A trimeric complex formed by Tub4p, the budding yeast γ-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5′ and 3′ cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3′ end. The deduced protein possesses 22% identity and 45% similarity with the yeast homologue. We further report that the human Spc98p, like γ-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both γ-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and γ-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction.     

A short C-terminal peptide in Gγ regulates Gβγ signaling efficacy

G protein beta-gamma (Gβγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM localization and functioning of Gβγ, allowing GPCR-G protein signaling to proceed. The diverse Gγ family has 12 members, and we have recently shown that the signaling efficacies of major Gβγ effectors are Gγ-type dependent. This dependency is due to the distinct series of membrane-interacting abilities of Gγ. However, the molecular process allowing for Gβγ subunits to exhibit a discrete and diverse range of Gγ-type–dependent membrane affinities is unclear and cannot be explained using only the type of prenylation. The present work explores the unique designs of membrane-interacting CT residues in Gγ as a major source for this Gγ-type–dependent Gβγ signaling. Despite the type of prenylation, the results show signaling efficacy at the PM, and associated cell behaviors of Gβγ are governed by crucially located specific amino acids in the five to six residue preprenylation region of Gγ. The provided molecular picture of Gγ–membrane interactions may explain how cells gain Gγ-type–dependent G protein-GPCR signaling as well as how Gβγ elicits selective signaling at various subcellular compartments.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Formation of α-Hydroxyglutaric Acid by Aspergillus glaucus

Aspergillus glaucus, cultured on sodium propionate-mineral salts medium, incorporates ¹⁴C-glyoxylate into labeled α-hydroxyglutaric acid within 30 sec. Mycelial extracts retain this biosynthetic capacity, which is destroyed by heating. Propionyl-2-¹⁴C-coenzyme A also in incorporated into labeled α-hydroxyglutaric acid by these mycelial extracts, but to a more limited extent. ¹⁴CO₂ evolution studies, employing differentially labeled ¹⁴C-propionate, indicate C-1 is oxidized by the mold before C-2, and C-2 before C-3. These findings suggest the involvement of α-hydroxyglutaric acid in the catabolism of propionic acid by A. glaucus.