The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By analogy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-activating enzyme. The second gene is SKP1, shown previously to be required for some ubiquitin-conjugation events. A deletion allele of ENR2 is lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conjugation to Cdc53p is required for optimal assembly or function of the E3 complex SCF Cdc4. Consistent with this model, both enr2⌬ and an allele of Cdc53p that is not Rub1p modified, render cells sensitive to alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.

Centromeres and several promoters of Saccharomyces cerevisiae contain a highly conserved octanucleotide, RTCACRTG, called CDEI. Using biochemical, genetic and structural analyses, we show that the same protein binds in vivo to CDEI sites in centromeres and in promoters. This protein, called CPF1 for centromere promoter factor, binds DNA as a dimer. Inactivation of the gene is not lethal but leads to a partial loss of the centromere function and to a Met‐ phenotype. Changes of the chromatin structure due to inactivation of CPF1 are seen at centromeres and at several CDEI‐carrying promoters (e.g. MET25, TRP1, GAL2). However promoter activities are affected in diverse ways making it presently difficult to describe a function for CPF1 in gene expression. The sequence of the cloned gene reveals in the carboxy‐terminal part two potential amphipathic helices preceded by a positively charged stretch of amino acids very similar to the helix‐loop‐helix domains recently identified in factors controlling tissue specific transcription in higher eukaryotes. Carboxy‐terminal truncations of CPF1 lacking this domain no longer bind to CDEI. The amino‐terminal half of CPF1 carries two clusters of negatively charged amino acid residues. Surprisingly, deletions of these clusters still render cells Met+ and lead only to a marginal decrease in centromere activity.

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability ofSaccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B. CBF5p A 240-kDa multisubunit protein complex (CBF3) that binds specifically to the CDEIII region of the yeast centromere has been purified and characterized (20). This protein complex is thought to be absolutely essential for centromere and kinetochore function, since it binds in vitro to the wild-type CEN sequence but not to a functionally inactive mutated CEN DNA that contains a single-base alteration in CDEIII (20,24). Affinity-purified CBF3 consists of at least three tightly associated subunits: 110 kDa (CBF3A), 64 kDa (CBF3B), and 58 kDa (CBF3C). Significantly, affinity-purified preparations of CBF3 contain an ATP-dependent motor activity that mediates movement of CEN DNA-coated microbeads along microtubules in a plus-to-minus direction (15). Thus, it is likely that CBF3 contains a kinetochore component that brings about attachment and movement of the chromosomes on the spindle microtubules. The gene (CBF2) encoding the 110-kDa subunit of this complex was cloned by using tryptic peptide sequences obtained from gel-purified CBF3A as a basis to prepare synthetic oligodeoxyribonucleotide hybridization probes, which were subsequently used to screen a yeast genomic library (17). At the same time, Goh and Kilmartin (12) cloned the identical gene by complementation of a conditional mutation (ndclO) leading to a defect in chromosome segregation. The CBF2/ NDC10 gene product is essential for viability of S. cerevisiae and for proper chromosome segregation. Although this protein contains a consensus nucleotide-binding site, no homologies to known molecular motors are apparent.In this paper, we describe the isolation of two yeast low-affinity centromere-binding proteins, topoisomerase II (Topo II) and CBF5p, and the cloning and sequencing of the CBF5 gene. The CBF5 protein contains a (KKD/E)n sequence domain highly homologous to known microtubulebinding domains in microtubule-associated protein 1A (MAP1A) and MAPlB (19,26). Evidence indicating that 4884 on May 12, 2018 by guest

Label-free bio-sensing is a critical functionality underlying a variety of health- and security-related applications. Micro-/nano-photonic devices are well suited for this purpose and have emerged as promising platforms in recent years. Here we propose and demonstrate an approach that utilizes the optical spring effect in a high-Q coherent optomechanical oscillator to dramatically enhance the sensing resolution by orders of magnitude compared with conventional approaches, allowing us to detect single bovine serum albumin proteins with a molecular weight of 66 kDa at a signal-to-noise ratio of 16.8. The unique optical spring sensing approach opens up a distinctive avenue that not only enables biomolecule sensing and recognition at individual level, but is also of great promise for broad physical sensing applications that rely on sensitive detection of optical cavity resonance shift to probe external physical parameters.

Abstract. We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present.The CBP-2 gene is essential for viability of yeast and is identical to NDCIO, in which a conditional mutation leads to a defect in chromosome segregation (Gob, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.