Direct analysis of protein complexes using mass spectrometry.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

Mating in Saccharomyces cerevisiae: the role of the pheromone signal transduction pathway in the chemotropic response to pheromone.

The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the beta or gamma subunit (ste4 and ste18) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage.

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9 delta, rad 17 delta, and rad24 delta. Furthermore, the triple rad9 delta rad17 delta rad24 delta mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1 delta, mag1 delta, apn1 delta, rev3 delta, rad18 delta, rad16 delta, dun1-delta 100, sad4-1, tel1 delta, rad26 delta, rad51 delta, rad52-1, rad54 delta, rad14 delta, rad1 delta, pol30-46, pol30-52, mad3 delta, pds1 delta/esp2 delta, pms1 delta, mlh1 delta, and msh2 delta are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.

Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint.

A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the double-strand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G2, these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.

Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae.

Two DNA sequences that reduce mitotic fidelity of chromosome transmission have been identified: MIF1 and MIF2. MIF1 is a unique sequence located on the right arm of chromosome XII that stimulates loss and recombination for both chromosomes V and VII when present in a high copy number plasmid. MIF1 is not essential for cell division but is necessary for the normal fidelity of chromosome transmission. MIF2 is a unique sequence located 15 cM distal to HIS6 on chromosome IX that induces a high frequency of chromosome VII loss and a lower frequency of chromosome V loss when present in high copy number; it has no effect on mitotic recombination. Disruption of the genomic MIF2 locus was lethal and cells lacking this function arrested division with a terminal phenotype characteristic of a block in DNA replication or nuclear division.

New cytoplasmic genetic element that controls 20S RNA synthesis during sporulation in yeast.

Under conditions that induce meiosis and sporulation in Saccharomyces cerevisiae, most strains accumulate a 20S RNA, amounting to as much as 15% of the newly synthesized RNA. The ability of cells to accumulate this new RNA species depends on a dominant genetic element that is cytoplasmically inherited, but is distinct from the other cytoplasmic elements that have been previously identified. The ability to synthesize 20S RNA does not depend on mitochondrial DNA, 2-micron DNA, the translational suppressor psi, the genetic element carrying URE3, or double-stranded killer RNA. However, all 20S- strains examined were also nonkillers, although many nonkiller strains were 20S+. This work also shows that 20S RNA accumulating is not essential for sporulation even though it is induced only by conditions that initiate sporulation. Furthermore, strains that are unable to complete meiosis are still capable of producing 20S RNA when placed under the nitrogen starvation conditions that promote sporulation.

A new gene affecting the efficiency of mating-type interconversions in homothallic strains of Saccharomyces cerevisiae.

Homothallic strains of Saccharomyes cerevisiae are able to switch efficiently from one mating genotype to another. From a single haploid spore arise both a and mating type cells, which then self-mate to produce a colony consisting almost exclusively of nonmating a/ diploid cells. We have isolated a mutant homothallic strain that gives rise to colonies that show bisexual mating behavior. The mating reaction is always asymmetric, that is, in some colonies a mating is much stronger than mating, while others show greater than a mating.-This mating phenotype arises from the presence of three cell types in a colony: some a/ nonmating diploids and an unequal number of a and haploid cells. The predominant haploid type is that of the original cell that gives rise to the colony. This mixture of cell types arises from a very reduced efficiency of homothallic mating-type interconversions in the mutant strain.-The mutation, designated switch (swi1-1), behaves as a single genetic locus. The mutation is centromere linked, but not linked to the mating type locus or to any of the homothallism genes: HO, HMa and HM. The switch mutation does not affect the efficiency of self-mating, but rather directly affects the frequency of interconversion of mating types.