Telomeres are the physical ends of eukaryotic chromosomes. Genetic studies have established that the baker's yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown. Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides. The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide. In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active. We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.

Genetic configurations resulting in high ratios of beta-tubulin to alpha-tubulin are toxic in S. cerevisiae, causing microtubule disassembly and cell death. We identified three non-tubulin yeast genes that, when overexpressed, rescue cells from excess beta-tubulin. One, RBL2, rescues beta-tubulin lethality as efficiently as does alpha-tubulin. Rbl2p binds to beta-tubulin in vivo. Deficiencies or excesses of either Rbl2p or alpha-tubulin affect microtubule-dependent functions in a parallel fashion. Rbl2p has functional homology with murine cofactor A, a protein important for in vitro assays of beta-tubulin folding. The results suggest that Rbl2p participates in microtubule morphogenesis but not in the assembled polymer.

No abstract

The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation.

We generated a strain of Saccharomyces cerevisiae in which the sole source of alpha-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S alpha-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15 degrees C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in alpha-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated alpha-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated alpha-tubulin in C377S tub1 cells. Our results suggest that cys-377 of alpha-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.

The yeast protein Rbl2p suppresses the deleterious effects of excess ␤-tubulin as efficiently as does ␣-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with ␤-tubulin that does not contain ␣-tubulin, thus defining a second pool of ␤-tubulin in the cell. Formation of the complex depends upon the conformation of ␤-tubulin. Newly synthesized ␤-tubulin can bind to Rbl2p before it binds to ␣-tubulin. Rbl2p can also bind ␤-tubulin from the ␣/␤-tubulin heterodimer, apparently by competing with ␣-tubulin. The Rbl2p-␤-tubulin complex has a half-life of ϳ2.5 h and is less stable than the ␣/␤-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing ␤-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p-␤-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.Much of the work on microtubules has focused on the assembly reaction from ␣/␤-tubulin heterodimer to polymer. This reaction is well characterized in vitro, and genetic and pharmacological studies demonstrate its importance and possible in vivo mechanisms for its regulation. Less well understood are the steps leading to the formation of the heterodimer in the cell. There is now considerable evidence that these steps are themselves subject to cellular controls crucial for microtubule function.The proper folding of the tubulin chains in vivo (22, 23) and in vitro (6,12,26) apparently requires the action of chaperone complexes (variously abbreviated as TriC/CCT/TCP/c-cpn). Unlike other proteins that are TriC substrates, however, ␣-and ␤-tubulin require other proteins in vitro to exchange into exogenous heterodimers, as assayed by native gel electrophoresis (2,7,8). The extent to which this in vitro reaction is applicable to the in vivo situation is unknown, beginning as it does with fully denatured protein rather than newly synthesized protein (5). Comparison of elements of the in vitro reaction with cellular activities reveals both similarities and differences. For example, yeast strains with altered forms of TCP-1 genes do exhibit cytoskeleton defects (3,15,(22)(23)(24). On the other hand, a protein that is required for the in vitro reaction is the homolog of a yeast protein, Cin1p, that is not essential in vivo but which may be involved in microtubule functions (10,20,21).A recent study of the in vitro folding reaction identified cofactor A, which promotes the recovery of ␤-tubulin, as a monomer from the chaperonin (7). However, in this assay, the form of ␤-tubulin released by cofactor A does not exchange into exogenous dimer. A genetic analysis of cellular responses to ␤-tubulin levels identified Rbl2p as a yeast structural homolog of cofactor A; Rbl2p is a nonessential protein that suppresses the lethality associated with overexpression of ␤-tubulin (1). The murine cofactor A was shown to partially replace Rbl2p in this in vivo assay (1). Although results of the in vitro assay first suggested that cofact...

This essay compares the approaches that scientific societies in the ACCESS meta-organization use to implement and assess travel award programs for URM trainees and presents a set of recommendations, including both short- and long-term outcomes assessment in populations of interest and specialized programmatic activities coupled to travel award programs.

extend along the major axis of the cell during interphase, then depolymerize to form the intranuclear spindle. Dis-Department of Biology ruption of microtubules, either by drugs or by mutations and Center for Cancer Research in tubulin genes, causes abnormal growth, bending, and