Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin.

In most eukaryotic cells, the C-terminal amino acid of alpha-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of alpha-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin-Tyr ligase. There is evidence that tubulin-Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

STOP proteins are responsible for the high degree of microtubule stabilization observed in neuronal cells.

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.

Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts.

A number of cycling mammalian cells, such as NIH 3T3, contain abundant subsets of cold-stable microtubules. The origin of such microtubule stabilization in nonneuronal cells is unknown. We have previously described a neuronal protein, stable tubule-only polypeptide (STOP), that binds to microtubules and induces cold stability. We find that NIH 3T3 fibroblasts contain a major 42-kDa isoform of STOP (fibroblastic STOP, F-STOP). F-STOP contains the central repeats characteristic of brain STOP but shows extensive deletions of N- and C-terminal protein domains that are present in brain STOP. These deletions arise from differences in STOP RNA splicing. Despite such deletions, F-STOP has full microtubule stabilizing activity. F-STOP accumulates on cold-stable microtubules of interphase arrays and is present on stable microtubules within the mitotic spindle of NIH 3T3 cells. STOP inhibition by microinjection of affinity-purified STOP central repeat antibodies into NIH 3T3 cells abolishes both interphase and spindle microtubule cold stability. Similar results were obtained with Rat2 cells. These results show that STOP proteins have nonneuronal isoforms that are responsible for the microtubule cold stability observed in mammalian fibroblasts.

Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins.

Pheromone response in yeast: association of Bem1p with proteins of the MAP kinase cascade and actin.

Haploid cells of the yeast Saccharomyces cerevisiae respond to mating pheromones with polarized growth toward the mating partner. This morphological response requires the function of the cell polarity establishment protein Bem1p. Immunochemical and two-hybrid protein interaction assays revealed that Bem1p interacts with two components of the pheromone-responsive mitogen-activated protein (MAP) kinase cascade, Ste20p and Ste5p, as well as with actin. Mutants of Bem1p that are associated with defective pheromone-induced polarized morphogenesis interacted with Ste5p and actin but not with Ste20p. Thus, the association of Bem1p with Ste20p and Ste5p may contribute to the conveyance of spatial information that regulates polarized rearrangement of the actin cytoskeleton during yeast mating.

Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p.

The mating response pathway of the yeast Saccharomyces cerevisiae includes a heterotrimeric guanine nucleotide-binding protein (G protein) that activates a mitogen-activated protein MAP kinase cascade by an unknown mechanism. An amino-terminal fragment of the MAP kinase scaffold protein Ste5p that interfered with pheromone-induced cell cycle arrest was identified. A haploid-specific interaction between the amino terminus of Ste5p and the G protein beta subunit Ste4p was also detected in a two-hybrid assay, and the product of a signaling-defective allele of STE4 was defective in this interaction. In cells with a constitutively activated pheromone response pathway, epitope-tagged Ste4p was coimmunoprecipitated with Ste5p. Thus, association of the G protein and the MAP kinase cassette via the scaffolding protein Ste5p may transmit the G protein signal.