Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain.

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 N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.

A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex.

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing 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.

A yeast TCP-1-like protein is required for actin function in vivo.

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 of cytosolic 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-type 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 enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo.

Selective expression of methotrexate-resistant dihydrofolate reductase (DHFR) activity in mice transduced with DHFR retrovirus and administered methotrexate.

To determine the effect of in vivo pharmacological selective pressure on the insertion and expression of new gene sequences, retroviral-mediated transfer of a methotrexate-resistant dihydrofolate reductase (Mtxr-DHFR) gene in hematopoietic tissue was investigated using a murine syngeneic bone marrow transplant system. A series of recombinant retroviral vectors were constructed to contain long terminal repeat (LTR) regions from different murine retroviruses associated with various proliferative disorders of the lymphohematopoietic system, including Moloney leukemia virus, spleen focus-forming virus (anemia strain) and myeloproliferative sarcoma virus. High-titer DHFR virus (10(7) colony-forming units/ml) was generated by gene amplification adapting virus-producer cell lines to grow in medium containing increasing concentrations of Mtx. This high-titer DHFR virus was used to introduce the Mtxr-DHFR gene into murine hematopoietic tissue by injecting DHFR virus-exposed marrow into lethally irradiated syngeneic recipient mice that subsequently were administered Mtx. Southern blot analysis of spleen DNA demonstrated insertion of the DHFR provirus in all surviving mice transplanted with DHFR virus-exposed marrow. However, enzymatic assay of crude spleen extracts demonstrated the presence of Mtxr-DHFR activity only in mice that were administered Mtx; nonadministered animals or animals transplanted with control (neo) virus-infected marrow contained undetectable drug-resistant enzyme activity. These results suggest the selective outgrowth of hematopoietic tissue harboring and expressing a DHFR provirus in animals administered Mtx and have implications for the application of drug-resistance gene insertion in somatic tissues of animals and humans.

Screens for extragenic mutations that fail to complement act1 alleles identify genes that are important for actin function in Saccharomyces cerevisiae.

Null mutations in SAC6 and ABP1, genes that encode actin-binding proteins, failed to complement the temperature-sensitive phenotype caused by a mutation in the ACT1 gene. To identify novel genes whose protein products interact with actin, mutations that fail to complement act1-1 or act1-4, two temperature-sensitive alleles of ACT1, were isolated. A total of 14 extragenic noncomplementing mutations and 12 new alleles of ACT1 were identified in two independent screens. The 14 extragenic noncomplementing mutations represent alleles of at least four different genes, ANC1, ANC2, ANC3 and ANC4 (Actin NonComplementing). Mutations in the ANC1 gene were shown to cause osmosensitivity and defects in actin organization; phenotypes that are similar to those caused by act1 mutations. We conclude that the ANC1 gene product plays an important role in actin cytoskeletal function. The 12 new alleles of ACT1 will be useful for further elucidation of the functions of actin in yeast.

Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae.

We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.

Assay for expression of methotrexate-resistant dihydrofolate reductase activity in the presence of drug-sensitive enzyme.

A simple, continuous spectrophotometric assay for dihydrofolate reductase (DHFR) activity was adapted for determination of drug-resistant enzyme activity expressed from transfected genes in cells containing drug-sensitive enzyme. Methotrexate inhibition characteristics in this assay system were assessed for the murine wild-type (WT) enzyme as well as variant genes encoding amino acid substitutions at codon positions 22 (arg22) or 31 (trp31) expressed in DHFR-deficient Chinese hamster ovary (CHO) cells and in mouse 3T3 cells. Methotrexate concentrations were thus identified which maximized inhibition of the wild-type enzyme while maintaining substantial arg22 or trp31 activity. Mixing experiments were conducted to determine the minimum amount of drug-resistant enzyme distinguishable from a constant amount of wild-type enzyme in the presence of methotrexate. Mixtures of enzymes from a variety of sources (WT, arg22, or trp31 expressed in CHO or 3T3 cells) demonstrated a detection limit of 0.03 to 0.06 nmol/min. Assay of methotrexate-resistant arg22 DHFR appeared to be limited by the low level of activity associated with this enzyme variant, whereas assay of the trp31 variant was limited by enzyme inhibition at lower concentrations of methotrexate. The assay was thus applicable to two quite diverse DHFR variants and may be useful for assaying the expression of other drug-resistant DHFR genes as well after introduction into cells containing drug-sensitive enzyme.