We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits ofcytosolc heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-tye Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enh the phenotypes of actin mutants. Taken together, these findin demonstrate that TCP-l-like proteins are required for actin and tubulin function in vivo.TCP-1 (t-complex polypeptide 1) is a subunit of eukaryotic cytosolic chaperoning. It has sequence homology to the subunits of bacterial (GroEL/hsp60), plastid, and mitochondrial (chaperonin 60) chaperonins (1, 2), protein complexes that facilitate protein folding in cells (3-5). The structure of the cytosolic chaperonin, a two-layered torus composed of multiple subunits (6)(7)(8), is similar to that of the bacterial, mitochondrial, and plastid chaperoning, but the subunit composition is more complex. Recent studies have identified distinct families of TCP-1-related proteins, referred to as CCT (chaperonin containing TCP-1) proteins (9). Within a family, the CCT proteins share -60%o sequence identity across species lines, while between families the identity is only =30%6, even within a single species such as mouse or yeast.TCP-1-containing chaperonins from rabbit, mouse, and bovine tissues can facilitate the folding of newly synthesized actin and tubulin (8, 10, 11) as well as actin-related and tubulin-related proteins (12). In addition, in CHO cell lysates, tubulin and a 43-kDa protein that comigrates with actin on polyacrylamide gels are bound to chaperonins (13 (20) to create pDV211. A nested deletion series was created from pDV211 by using Exo III and was used for sequencing the sense strand ofANC2 (nt 1-2727; Biomolecular Resource Center sequencing facility, University of California, San Francisco). The antisense strand of ANC2 was sequenced from nt 2307-439 by using oligonucleotide primers.ANC2 Deletion and Genetic Linkage Analysis. The ANC2 null allele was generated by replacing nt 530-2305 (EcoNI/ Hpa I fragment), which include the entire chromosomal ANC2 coding sequence plus 194 bp upstream, in two different wild-type diploid strains (DDY426 and DDY665) with the integrating plasmid pDV219, which contains the LEU2 gene with ANC2 flanking sequences at the 5' (BamHI/EcoNI; 530 bp) and the 3' (Hpa I/BamHI; 435 bp) ends. A total of 60 tetrads derived from six independent transformants for both parental strains ...

Nucleation of microtubules is central to assembly of the mitotic spindle, which is required for each cell division. ␥-Tubulin is a universal component essential for microtubule nucleation from centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast ␥-tubulin complex (Tub4p complex) produced in insect cells. The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle pole body docking protein Spc110p affects its nucleating activity. The solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the K d for binding is 150 nM. The low nucleation activity of the Tub4p complex is not enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components or modifications to achieve robust activity. Finally, we report the identification of a large 22 S Tub4p complex in yeast extract that contains multimers of Spc97p similar to ␥-tubulin ring complexes found in higher eukaryotic cells.

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubuleorganizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110 -221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98 -63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98 -63 spc110 -221 cells is caused by the failure of Spc98 -63p to interact with Spc110 -221p. In contrast, the lethal phenotype in spc97-62 spc110 -221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser 60 , Thr 64 , and Thr 68 are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser 36 , is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser 36 are located within the Nterminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.

P-bodies (PB) are ribonucleoprotein (RNP) complexes that aggregate into cytoplasmic foci when cells are exposed to stress. While the conserved mRNA decay and translational repression machineries are known components of PB, how and why cells assemble RNP complexes into large foci remain unclear. Using mass spectrometry to analyze proteins immunoisolated with the core PB protein Dhh1, we show that a considerable number of proteins contain low-complexity (LC) sequences, similar to proteins highly represented in mammalian RNP granules. We also show that the Hsp40 chaperone Ydj1, which contains an LC domain and controls prion protein aggregation, is required for the formation of Dhh1-GFP foci upon glucose depletion. New classes of proteins that reproducibly co-enrich with Dhh1-GFP during PB induction include proteins involved in nucleotide or amino acid metabolism, glycolysis, tRNA aminoacylation, and protein folding. Many of these proteins have been shown to form foci in response to other stresses. Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins, suggesting an active role for RNA in PB function. Thus, global characterization of PB composition has uncovered proteins and RNA that are important for PB assembly.