Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication

The 42-kD component of the S. cerevisiae spindle pole body (SPB) localizes to the electron-dense central plaque of the SPB. We have cloned the corresponding gene SPC42 (spindle pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiled-coil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential in normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a disc- or dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane.     

Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.     

Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation.

The yeast microtubule organizing centre (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules which are functionally and spatially distinct. Microtubule organization requires the yeast gamma-tubulin complex (Tub4p complex) which binds to the nuclear side of the SPB at the N-terminal domain of Spc110p. Here, we describe the identification of the essential SPB component Spc72p whose N-terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. We further report that this Tub4p complex-binding domain of Spc72p is essential and that temperature-sensitive alleles of SPC72 or overexpression of a binding domain-deleted variant of SPC72 (DeltaN-SPC72) impair cytoplasmic microtubule formation. Consequently, polynucleated and anucleated cells accumulated in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules compared with wild-type. Finally, exchange of the Tub4p complex-binding domains of Spc110p and Spc72p established that the Spc110p domain, when attached to DeltaN-Spc72p, was functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p fused to DeltaN-Spc110p led to a dominant-negative effect. These results suggest that different components of MTOCs act as receptors for gamma-tubulin complexes and that they are essential for the function of MTOCs.     

Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature- sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature- sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.     

A Mutational Analysis Identifies Three Functional Regions of the Spindle Pole Component Spc110p in Saccharomyces cerevisiae

The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the γ-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. Our results strongly argue that Spc110p links the γ-tubulin complex to the central plaque of the SPB.     

Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe.

In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB). The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells. Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S. pombe) and the filamentous fungus Aspergillus nidulans. Like Spc110p and kendrin, Pcp1p and the A. nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region. Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy. Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.     

The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication.

Previously, we have shown that the gamma-tubulin Tub4p and the spindle pole body component Spc98p are involved in microtubule organization by the yeast microtubule organizing centre, the spindle pole body (SPB). In this paper we report the identification of SPC97 encoding an essential SPB component that is in association with the SPB substructures that organize the cytoplasmic and nuclear microtubules. Evidence is provided for a physical and functional interaction between Tub4p, Spc98p and Spc97p: first, temperature-sensitive spc97(ts) mutants are suppressed by high gene dosage of SPC98 or TUB4. Second, Spc97p interacts with Spc98p and Tub4p in the two-hybrid system. Finally, immunoprecipitation and fractionation studies revealed complexes containing Tub4p, Spc98p and Spc97p. Further support for a direct interaction of Tub4p, Spc98p and Spc97p comes from the toxicity of strong SPC97 overexpression which is suppressed by co-overexpression of TUB4 or SPC98. Analysis of temperature-sensitive spc97(ts) alleles revealed multiple spindle defects. While spc97-14 cells are either impaired in SPB separation or mitotic spindle formation, spc97-20 cells show an additional defect in SPB duplication. We discuss a model in which the Tub4p-Spc98p-Spc97p complex is part of the microtubule attachment site at the SPB.     

Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body.

NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.     

A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-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.     

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.     

Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p.

Previously, we have shown that the yeast gamma-tubulin, Tub4p, forms a 6S complex with the spindle pole body components Spc98p and Spc97p. In this paper we report the purification of the Tub4p complex. It contained one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. We addressed how the Tub4p complex binds to the yeast microtubule organizing center, the spindle pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p of the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. Finally, we isolated a complex containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. We discuss in a model, how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB.     

Microtubule organization by the budding yeast spindle pole body.

In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.     

Analysis of coiled-coil interactions between core proteins of the spindle pole body

The spindle pole body (SPB) is a multiprotein complex that organizes microtubules in yeast. Due to its large size and association with the nuclear membrane, little is known about its detailed structure. In particular, although many SPB components and some of the interactions between them have been identified, the molecular details of how most of these interactions occur are not known. The prevalence of predicted coiled-coil regions in SPB proteins suggests that some interactions may occur via coiled coils. Here this hypothesis is supported by biochemical characterization of isolated coiled-coil peptides derived from SPB proteins. Formation of four strongly self-associating coiled-coil complexes from Spc29, Spc42, and Spc72 was demonstrated by circular dichroism (CD) spectroscopy and a fluorescence resonance energy transfer (FRET) assay. Many weaker self- and heteroassociations were also detected by CD, FRET, and/or cross-linking. The thermal stabilities of nine candidate homooligomers were assessed; six unfolded cooperatively with melting temperatures ranging from <11 to >50 degrees C. Solution studies established that coiled-coil peptides derived from Spc42 and Spc72 form parallel dimers, and this was confirmed for Spc42 by a high-resolution crystal structure. These data contribute to a growing body of knowledge that will ultimately provide a detailed model of the SPB structure.     

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.     

The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.     

The nuclear pore complex-associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly

The two yeast proteins Mlp1p and Mlp2p (homologues of the vertebrate protein Tpr) are filamentous proteins attached to the nuclear face of nuclear pore complexes. Here we perform a proteomic analysis, which reveals that the two Mlps have strikingly different interacting partners, testifying to their different roles within the cell. We find that Mlp2p binds directly to Spc110p, Spc42p, and Spc29p, which are three core components of the spindle pole body (SPB), the nuclear envelope-associated yeast spindle organizer. We further show that SPB function is compromised in mlp2 mutants. Cells lacking Mlp2p form significantly smaller SPBs, accumulate aberrant SPB component-containing structures inside the nucleus, and have stochastic failures of cell division. In addition, depletion of Mlp2p is synthetically lethal with mutants impaired in SPB assembly. Based on these data, we propose that Mlp2p links the SPB to the peripheral Mlp assembly, and that this linkage is required for efficient incorporation of components into the SPB.     

The 110-kD spindle pole body component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent manner

Spc110p (Nuf1p) is an essential component of the yeast microtubule organizing center, or spindle pole body (SPB). Asynchronous wild-type cultures contain two electrophoretically distinct isoforms of Spc110p as detected by Western blot analysis, suggesting that Spc110p is modified in vivo. Both isoforms incorporate 32Pi in vivo, suggesting that Spc110p is post-translationally modified by phosphorylation. The slower-migrating 120-kD Spc110p isoform after incubation is converted to the faster-migrating 112-kD isoform after incubation with protein phosphatase PP2A, and specific PP2A inhibitors block this conversion. Thus, additional phosphorylation of Spc110p at serine and/or threonine residues gives rise to the slower-migrating 120-kD isoform. The 120-kD isoform predominates in cells arrested in mitosis by the addition of nocodazole. However, the 120-kD isoform is not detectable in cells grown to stationary phase (G0) or in cells arrested in G1 by the addition of alpha-factor. Temperature-sensitive cell division cycle (cdc) mutations demonstrate that the presence of the 120-kD isoform correlates with mitotic spindle formation but not with SPB duplication. In a synchronous wild-type population, the additional serine/threonine phosphorylation that gives rise to the 120-kD isoform appears as cells are forming the mitotic spindle and diminishes as cells enter anaphase. None of several sequences similar to the consensus for phosphorylation by the Cdc28p (cdc2p34) kinase is important for these mitosis-specific phosphorylations or for function. Carboxy-terminal Spc110p truncations lacking the calmodulin binding site can support growth and are also phosphorylated in a cell cycle-specific manner. Further truncation of the Spc110p carboxy terminus results in mutant proteins that are unable to support growth and now migrate as single species. Collectively, these results provide the first evidence of a structural component of the SPB that is phosphorylated during spindle formation and dephosphorylated as cells enter anaphase.     

The Bbp1p-Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication

In budding yeast, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope via its central plaque structure. Here, we describe the identification of BBP1 in a suppressor screen with a conditional lethal allele of SPC29. Bbp1p was detected at the central plaque periphery of the SPB and bbp1-1 cells were found to be defective in SPB duplication. bbp1-1 cells extend their satellite into a duplication plaque like wild-type cells; however, this duplication plaque then fails to insert properly into the nuclear envelope and does not assemble a functional inner plaque. This function in SPB duplication is probably fulfilled by a stable complex of Bbp1p and Mps2p, a nuclear envelope protein that is also essential for duplication plaque insertion. In addition, we found that Bbp1p interacts with Spc29p and the half-bridge component Kar1p. These interactions are likely to play a role in connecting the SPB with the nuclear envelope and the central plaque with the half-bridge.     

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.