Selective protein kinase inhibitors were developed on the basis of unexpected binding mode 2,6,9-trisubstituted purines to adenosine triphosphate–binding site human cyclin-dependent 2 (CDK2). By iterating chemical library synthesis and biological screening, potent CDK2–cyclin A complex Saccharomyces cerevisiae Cdc28p identified. The structural for affinity selectivity was determined by analysis a three-dimensional crystal structure CDK2-inhibitor complex. cellular effects these compounds...

A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of digested with a proteolytic enzyme produce large collection peptides. The complex peptide then separated on-line spectrometer, acquiring numbers spectra. spectra are used search protein database present. Results from standard show that present simple can be readily identified...

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p an abundant component the cortical cytoskeleton binds to F-actin with high (Kd 6 × 10−9 M). promotes rapid barbed-end assembly filaments cross-links into bundles more complex networks, but does not stabilize them. Genetic analyses crn1Δ...

How kinetochore proteins are organized to connect chromosomes spindle microtubules, and whether any structural organizational themes common kinetochores from distantly related organisms, key unanswered questions. Here, we used affinity chromatography mass spectrometry generate a map of protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, H4, the histone H3-like CENP-A Cse4p, strongly suggesting that Cse4p replaces H3 in specialized...

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been focus of many recent studies. Here, we identify a novel mode Arp2/3 regulation mediated by highly conserved actin binding coronin. Yeast coronin (Crn1) physically associates with and inhibits WA- Abp1-activated nucleation in vitro. The inhibition occurs specifically absence preformed filaments, suggesting that Crn1 may restrict activity to sides filaments. inhibitory resides its coiled coil domain....

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated new activator, Abp1p, which associates tightly yeast Abp1p contains two acidic sequences (DDW) similar those found SCAR/WASp proteins. demonstrate that mutation these abolishes activation vitro. Genetic studies...

Dam1p, Duo1p, and Dad1p can associate with each other physically are required for both spindle integrity kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Spc19p, Spc34p, the previously uncharacterized proteins Dad2p Ask1p. This Dam1p appears to be regulated through phosphorylation multiple subunits at least one event changing during cell cycle. We also find that purified binds directly microtubules vitro affinity...

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. associates directly with the protein kinase these two proteins colocalize to mitotic spindle. show here stimulates vitro, likely vivo, activity of Ipl1, facilitates association The Ipl1-binding -stimulating activities both reside within a region containing homology metazoan inner centromere (INCENP). also bind Dam1, microtubule-binding spindle integrity kinetochore function. Dam1...

Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection duo1 dam1 alleles used to develop deeper understanding functions interactions Dam1p. Based on similarity mutant phenotypes, genetic between alleles, interdependent localization mitotic spindle, Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these indicated that perform shared function vivo components complex. are not required...

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins are either membrane bound or in cellular compartments topologically outside the cell from those remain soluble cytoplasm. This type occurs stepwise, with core oligosaccharide added endoplasmic reticulum and subsequent modifications occurring golgi. We used tunicamycin, an inhibitor one earliest steps synthesis N-linked oligosaccharide, to select for mutants resistant this antibiotic. Genetic,...

Although there has been a recent explosion in the identification of budding yeast kinetochore components, physical interactions that underlie function remain obscure. To better understand how kinetochores attach to microtubules and this attachment is regulated, we sought characterize among proteins, especially with respect microtubule-binding Dam1 complex. The complex plays crucial role chromosome-spindle key target for phospho-regulation by Aurora kinase Ipl1p. identify protein–protein...

In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair. We have established yeast a connection between activity damage repair, commitment to replication. generated double-stranded breaks cells vivo by expressing restriction endonuclease shown that mutants lacking p70 died while isogenic wild-type did not. Moreover, we discovered occurs spontaneously during normal mitotic growth, functions repair of this damage. also observed mitotically growing...

Mitotic chromosomes segregate at the ends of shortening spindle microtubules (MTs). In budding yeast, Dam1 multiprotein complex supports this dynamic attachment, thereby contributing to accurate chromosome segregation. Purified will track end a depolymerizing MT and can couple it microbead transport in vitro . The processivity such motions has been thought depend on rings that form around MTs, but possibility alternative coupling geometries contribute these motilities not considered. Here,...

Conditions were established for the self-assembly of milligram amounts purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin not stabilized by taxol; hybrid microtubules containing substoichiometric bovine stabilized. Yeast microtubule-associated proteins (MAPs) identified on affinity matrices made from and all-bovine microtubules. About 25 MAPs isolated. The amino-terminal sequences several these determined: three known metabolic enzymes, two GTP-binding...

The mitotic spindle consists of a complex network proteins that segregates chromosomes in eukaryotes. To strengthen our understanding the molecular composition, organization, and regulation spindle, we performed system-wide two-hybrid screen on 94 implicated function Saccharomyces cerevisiae. We report 604 predominantly novel interactions were detected multiple screens, involving 303 distinct prey proteins. uncovered pattern extensive between reflecting intricate organization spindle....

Accurate chromosome segregation during mitotic division of budding yeast depends on the multiprotein kinetochore complex, Dam1 (also known as DASH). Purified heterodecamers encircle microtubules (MTs) to form rings that can function "couplers," molecular devices transduce energy from MT disassembly into motion a cargo. Here we show depolymerization develops force against ring is sixfold larger than exerted coupler binds only one side an MT. Wild-type slow fourfold, but include mutant Dam1p...

The mitotic spindle is a complex and dynamic structure. Although much has been learned about how spindles assemble mediate chromosome segregation, rapidly irreversibly disassemble during telophase less clear. We used synthetic lethal screens in budding yeast to identify mutants defective disassembly. Real-time, live cell imaging analysis of disassembly was performed on nine this process. Results suggest that achieved by mechanistically distinct but functionally overlapping subprocesses:...