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Vol. 17, Issue 4, 1959-1970, April 2006
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* The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom;
Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347
Submitted July 22, 2005;
Revised January 17, 2006;
Accepted January 18, 2006
Monitoring Editor: Trisha Davis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Even though the SPB and the centrosome are morphologically distinct, they share a number of homologous structural components and regulators critical for function (for reviews, see Jaspersen and Winey, 2004).
The SPB is embedded in the nuclear envelope throughout the cell cycle (reviewed in Adams and Kilmartin, 2000; Jaspersen and Winey, 2004). As viewed by electron microscopy, the core SPB appears as three disklike layers: an outer plaque and an inner plaque on the cytoplasmic and nuclear side of the nuclear envelope, respectively, and a central plaque that spans the nuclear membrane (O'Toole et al., 1999). SPB duplication involves several distinct steps (Adams and Kilmartin, 1999; Jaspersen and Winey, 2004). It begins in G1 with the deposition of amorphous satellite materials containing several SPB components at the distal cytoplasmic tip of the half-bridge, a modified nuclear membrane structure associated with one side of the SPB. As SPB duplication continues, the satellite grows in size to form the duplication plaque that is composed of outer and central plaque components (Adams and Kilmartin, 1999). Insertion of the duplication plaque into the nuclear envelope and assembly of inner plaque components complete SPB duplication. The two SPBs are connected by a full-bridge, which is severed in half as the SPBs separate into each pole of the dividing cell.
The SPB components and their relative position within the organelle were identified using various genetic, biochemical, cytological, and proteomic approaches (for review see Jaspersen and Winey, 2004; Muller et al., 2005). Interestingly, none of the components of the core SPB are integral membrane proteins, raising the question of how the SPB is inserted and tethered in the nuclear envelope. A likely possibility is that one or more of the four integral membrane proteins known to localize to the SPB (Ndc1p, Mps2p, Mps3p, and Kar1p) are involved. Of these four proteins, Ndc1p and Mps2p have been implicated in SPB insertion after the formation of the duplication plaque based on analysis of mutant phenotypes (Winey et al., 1991, 1993). Both proteins localize to the SPB periphery and the central plaque region (Chial et al., 1998; Munoz-Centeno et al., 1999), making them good candidates to form a pore in the nuclear envelope into which the SPB is inserted. Mps2p has previously been shown to bind to Bbp1p, a soluble protein that also binds to the central plaque component Spc29p and to the half-bridge protein Kar1p (Schramm et al., 2000). Thus, Bbp1p is thought to be a protein that connects the core SPB to the nuclear envelope (Schramm et al., 2000). However, it is not known with which protein(s) Ndc1p associates at the SPB to facilitate SPB insertion or how Ndc1p and Mps2p interact. Cells carrying mutations in NDC1 (ndc1-1 and ndc1-39), MPS2 (mps2-1), or BBP1 (bbp1-1) result in duplication plaque that fails to insert into the nuclear envelope (Winey et al., 1991; Winey et al., 1993; Schramm et al., 2000; Lau et al., 2004), but it is not clear if these genes cooperate in one pathway leading to SPB insertion or if they define two partially redundant pathways.
In this study, we searched for NDC1 interacting partner(s) using the ndc1-39 allele in a dosage suppressor screen and identified NBP1 (Nap1-binding protein 1; Shimizu et al., 2000). NBP1 also shows genetic interactions with several other SPB components including MPS2, and we showed that Nbp1p binds to Ndc1p and Mps2p in a two-hybrid assay. Nbp1p is also found to be a component of the Mps2p-Bbp1p complex. By immunoelectron microscopy, Nbp1p localizes to the SPB periphery and to the central plaque of the SPB, where Ndc1p and Mps2p also reside. Consistent with a possible function at the SPB, we found that a "degron" allele of NBP1, nbp1-td, causes defects in SPB duplication. Together, our data support a model that Nbp1p is a SPB component that serves as a molecular linker between the core SPB and the nuclear envelope membrane proteins Ndc1p and Mps2p.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Yeast cells were grown in YPD media (1% yeast extract, 2% bactopeptone, and 2% glucose), or synthetic media with 2% glucose and supplemented with the appropriate amino acids. Plates containing 5-fluoroorotic acid (5-FOA) were made as previously described (Boeke et al., 1987).
Polymerase chain reaction (PCR)-based gene deletion (Longtine et al., 1998) was used to create strain 3445. PCR-based gene tagging (Longtine et al., 1998) was used to create strains 3326, 3322, and 3310. Others genes were epitope tagged at the endogenous loci using PCR-based methods (Knop et al., 1999). nbp1-td cells were constructed and grown as described (Labib et al., 2000; Kanemaki et al., 2003). Briefly, cells were synchronized in G1-phase of the cell cycle with 10 µg/ml 
-factor in YPA medium (1% yeast extract, 2% peptone, and 0.01% adenine) containing 3% raffinose (YPA-Raf) and 0.1 mM CuSO4 at 23°C. To induce degradation of Nbp1p-td, expression of UBR1 was induced by transferring cells to YP medium with 3% raffinose and 2% galactose (YPA-Raf/Gal) for 45 min at 23°C in the presence of 10 µg/ml -factor. Cells were then incubated for 50 min at 37°C before removing -factor by washing cells with YPA-Raf/Gal at 37°C (t = 0). Cells were continuously incubated in YPA-Raf/Gal at 37°C.
Plasmids and Nucleic Acid Techniques
DNA was manipulated using standard techniques as described (Ausubel et al., 1998). Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). PCR products were purified using the QIAEXII Gel Extraction Kit (Qiagen). Plasmids and PCR products were transformed into yeast strains using EZ Transformation Kit (Zymo Research, Orange, CA).
Two-micron URA3 plasmid containing +NBP1/+YLR458W was made by PCR-amplifying YLR458W and the NBP1 regions with primers NBP1-5'KpnI (5'-CGG GGT ACC GGT CAT TTC ACT TAA TGG-3'; KpnI site is underlined) and NBP1-3'XhoI (5'-CCG CTC GAG CGG GCG TCC ATA GTA AAG ATG-3'; XhoI site is underlined). Then, the PCR product was cut with SnaBI-EaeI, and ligated into the XhoI-NotI sites of pRS202 with the use of the XhoI-EcoRV DNA oligonucleotide adaptors (EZ Clone Systems, New Orleans, LA). The CEN-URA3 plasmid containing +NBP1/+YLR458W was created by cutting the PCR product mentioned above with XhoI-SnaBI and ligated into the SalI-SmaI sites of pALR10 (a gift from Alain Camasses). The CEN-TRP1 plasmid containing +NBP1/+YLR458W was generated by cutting the PCR product mentioned above with SnaBI-EaeI and was cloned into the SmaI-NotI sites of pRS314.
Two-micron URA3 and CEN-TRP1 plasmids containing +NBP1/-YLR458W, -NBP1/+YLR458W, and -NBP1/-YLR458W were made by site-directed mutagenesis (Kunkel et al., 1987, and according to the pGEM Single Strand Systems Manual [Promega, Madison, WI]; also see Lau et al., 2004). Single-stranded CEN-TRP1 plasmid (pRS314) containing +NBP1/+YLR458W was isolated from repair-incompetent bacteria CJ236 that has been infected with helper phage R408 (Promega). Then, primers A (GAT CTC TTC TTC AAA ATA CGC GTC ATA TCG CCA CAA ACA TTT TG; MluI site is underlined) and B (CAT CTC TGA TTG TCT GAA AGA TCT TTG TTA ACC TTC; BglII site is underlined) were added for in vitro second-strand synthesis to generate plasmid +NBP1/-YLR458W. Likewise, primers C (CGG ATT TTA GTA TAC TAC CAA TGT CTA TTA TTG; Bst1107I/BstZ17I site is underlined) and D (GTA TTC CAA AAA AAA GCT TTC ACA GTC CCT G; HindIII site is underlined) were used to generate plasmid -NBP1/+YLR458W, and primers A, C, and D were used to generate plasmid -NBP1/-YLR458W. The three plasmids were amplified, and the mutations generated were verified by the presence of the restriction site indicated and also by DNA sequencing. The XhoI-SacI fragments from these plasmids were cloned into the XhoI-SacI sites of pRS202 for further analysis.
Fluorescence Microscopy
Cells containing NBP1-GFP; SPC42-CFP and NBP1-GFP; NDC1-CFP were resuspended in PBSA without fixation. GFP and CFP signals were detected by their autofluorescence, and DNA was stained with 4',6-diamidono-2-phenylindole (DAPI; Sigma Chemical Co., St. Louis, MO). The cells were visualized using a Zeiss Axioplan 2 deconvolution fluorescence microscope (Thornwood, NY) equipped with a Cooke SensiCam CCD camera (Romulus, MI). The images were processed using SlideBook software (v. 3.0.11, Intelligent Imaging Innovations, Denver, CO).
CFP-, eqFP-, GFP-, and YFP-labeled cells were analyzed by fluorescence microscopy after fixation of cells with 4% paraformaldehyde (150 mM phosphate buffer, pH 6.5) for 10 min at 20°C. For a series of z-focal planes images were collected on a Zeiss Axiophot microscope using a Coolsnap HQ camera (Photometrics, Tucson, AZ) and Metamorph software (Universal Imaging, West Chester, PA). Images in different z-planes were projected and processed in Adobe Photoshop (San Jose, CA).
Electron Microscopy
Nbp1p-GFP cells were high-pressure frozen, freeze-substituted, sectioned, and stained for electron microscopy or for immunoelectron microscopy as previously described (Giddings et al., 2001; Lau et al., 2004). Serial thin sections were viewed on a Philips CM10 electron microscope (Mahwah, NJ), and images were captured with Gatan digital camera and viewed with Digital Micrograph software (Gatan, Pleasanton, CA). Some cells were glutaraldehyde-fixed and embedded into Spurr resin and then prepared for serial section electron microscopy (Byers and Goetsch, 1975).
Two-Hybrid Assay
GAL4 DNA-binding domain and GAL4 DNA-activation domain constructs containing NBP1, NDC1, and ndc1-39 were created by amplified as NcoI-XhoI fragments and inserted into the NcoI-SalI sites of pOBD2 or pOAD1 (Uetz et al., 2000). The remaining two-hybrid plasmids were gifts of Stan Fields, and their construction is described in Uetz et al. (2000). Plasmids were transformed into PJ69-4a for pOAD1 constructs, or PJ69-4 for pOBD2 constructs (James et al., 1996). Two-hybrid interactions were tested as described (Uetz et al., 2000).
Immunoprecipitation
Cells (80 OD600) were lysed with glass beads in L-buffer (50 mM Tris-HCl, pH 7.6, 10 mM EDTA, 1 mM EGTA, 100 mM NaCl, 5% glycerol) in the presence of complete protease inhibition cocktail (Roche Diagnostics, Branchburg, NJ). The lysates were then incubated with 1% Triton X-100 for 30 min at 4°C. The cell extracts were clarified by two consecutive centrifugations (20,000 x g, 5 min). The supernatants were incubated with IgG-Sepharose for 2 h at 4°C with rotation. Beads were washed three times with L-buffer, 1% Triton X-100 and once with TEV cleavage buffer (50 mM HEPES-KOH, pH 7.9, 150 mM KOAc, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT). Proteins were eluate using TEV protease. Samples were analyzed by immunoblotting with the indicated antibodies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) were screened for their ability to rescue the growth defects of ndc1-39 temperature-sensitive (ts) cells (Lau et al., 2004). Two clones (suppressors 140 and 107) that exhibited the strongest suppression phenotype were isolated and re-transformed into the ndc1-39 strain for further characterization. Suppressor 140 contained four complete open reading frames (ORFs) including MPS2, which encodes an integral membrane protein required for SPB duplication like Ndc1p (Munoz-Centeno et al., 1999; Schramm et al., 2000). Both, the suppressor 140 and MPS2, partially suppress the ts phenotype of ndc1-39 mutants at the restrictive temperature of 33.5°C, although not as well as a plasmid containing wild-type NDC1 (Figure 1A). The other ORFs on suppressor 140 besides MPS2 do not suppress ndc1-39 (unpublished observation). This is the first evidence of a genetic interaction between NDC1 and MPS2.
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Suppression of the growth defect of ndc1-39 cells by NBP1 suggests a functional interaction of both genes. To strengthen this conclusion, we examined nbp1-1 cells, which carry a R180W missense mutation in NBP1 (Shimizu et al., 2000) for genetic interactions with mutations in NDC1. Cells containing nbp1-1 exhibit severe growth defects compared with wild-type cells at 38°C (Figure 1D; Shimizu et al., 2000). We reasoned that if NBP1 functions with NDC1, then mutants containing mutations in two of these genes may have synergistic growth defects. Indeed, cells carrying both nbp1-1 and ndc1-39 were unable to grow at 30°C (Figure 1D), a temperature that was permissive for either of the individual mutations. Thus, nbp1-1 cells are synthetically sick in the presence of ndc1-39, further suggesting that NBP1 and NDC1 function together in the cell.
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NBP1 Also Suppresses the Growth Defect of Several Mutants Defective in SPB Duplication
The fact that both MPS2 and NBP1 suppress the growth defect of the ndc1-39 mutants led us to speculate that NBP1 might also interact with MPS2. Therefore, we tested NBP1 suppression on mps2-1 ts mutants using the constructs described above. Two-micron plasmids specifically containing wild-type NBP1 suppressed the mps2-1 ts phenotype as well as wild-type MPS2 at the semirestrictive temperature of 35°C (Figure 2). Intrigued by the ability of increased levels of NBP1 to suppress the ndc1-39 or the mps2-1 mutations, we asked whether NBP1 acts as a dosage suppressor of other known SPB duplication mutations. We found that NBP1 partially suppressed the growth defect of a subset of these mutants, namely spc42-11, cdc31-2, mps1-8, and mps3-1 (but not bbp1-1, cmd1-1, cmn67
, kar117, mps1-1, mps1-737, ndc1-1, nud1-44, spc29-2, spc29-3, spc29-20, spc42-10, spc98-2, spc110-220, tub4-1; unpublished observation). The results suggest that NBP1 is involved in some aspect of SPB function and/or duplication.
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NBP1 Is an Essential Gene
Nbp1p was originally isolated as a two-hybrid binding partner with Nap1p (Nucleosome Assembly Protein 1; Shimizu et al., 2000). NBP1 encodes a 319-amino acid protein that contains a coiled-coil domain and several potential phosphorylation sites for Cdc28p cyclin-dependent kinase and cAMP-dependent kinase (Shimizu et al., 2000), but lacks significant homology to any characterized protein. Although the authors suggest Nbp1p is required for G2/M transition, the molecular function of Nbp1p remains undefined.
Shimizu et al. (2000) also showed that disruption of the NBP1 region is lethal to yeast cells. However, eliminating the NBP1 ORF also deletes the overlapping ORF YLR458W (Figure 1B). To determine which of the two genes is essential for growth, we tested the ability of plasmids containing NBP1 and/or YLR458W start codon mutations to rescue growth in an nbp1/ylr458w strain. As expected, the nbp1/ylr458w strain with vector alone is not viable, whereas plasmid containing both NBP1 and YLR458W was able to rescue growth (Figure 3). A plasmid lacking NBP1 either in the presence or absence of YLR458W was unable to provide the essential function for growth (Figure 3). Only when the cells were provided with wild-type NBP1 was the lethality of the nbp1/ylr458w strain rescued (Figure 3). These results clearly show that NBP1 is an essential gene in yeast, similar to nearly all genes encoding SPB components and regulators.
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) and colocalizes with the SPB component Spc29p (Sundin et al., 2004). This localization is consistent with an association of Nbp1p either with SPBs or with kinetochores, which in yeast are clustered close to SPBs. Moreover, Ndc1p localizes to both SPBs and nuclear pore complexes (NPCs, Chial et al., 1998). Therefore, we also examined the possibility of Nbp1p localizing to the NPCs using deconvolution fluorescence microscopy. The endogenous copy of NBP1 was fused to green fluorescent protein (GFP), and the strain also contained an allele of the core SPB component Spc42p fused to cyan fluorescent protein (CFP). We consistently found that the number of Nbp1p-GFP spots observed was identical to the number of SPBs expected at the given cell cycle stage; in that one spot in unbudded G1 cells, one or two spots in small budded S-phase cells, and two spots in large budded mitotic cells (Figure 4A). In all of these cells, Nbp1p-GFP colocalized with Spc42p-CFP. This was also the case in metaphase cells when kinetochore proteins show a slightly distinct distribution from SPB components (e.g., Janke et al., 2002). Thus, Nbp1p is associated with the SPB.
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In addition, we fused Ndc1p with CFP in the Nbp1p-GFP strain, and we found that Nbp1p-GFP also colocalized with the Ndc1p-CFP at the SPBs, but it did not colocalize with Ndc1p to the nuclear periphery at NPCs (Figure 4B), suggesting that Nbp1p functions at the SPB and not with Ndc1p at the NPC.
To confirm the localization of Nbp1p to the SPB, a strain containing Nbp1p-GFP was examined by immunoelectron microscopy using anti-GFP primary antibodies and secondary antibodies conjugated to colloidal gold. Detailed examination of the collodial gold particles that decorated 11 SPBs revealed that about half of the Nbp1p-GFP signal was at the periphery of the SPB, one quarter was at the central plaque, and a few were at the nuclear envelope or at the outer plaque (Figure 4C). Consistent with our fluorescence microscopy analysis, Nbp1p-GFP was not observed elsewhere in the cell away from the SPB. These data demonstrate that Nbp1p is a SPB component predominantly found at the SPB periphery and the central plaque similar to the localization of Mps2p and Ndc1p at the SPBs (Chial et al., 1998; Munoz-Centeno et al., 1999).
Nbp1p Binds to Both Ndc1p and Mps2p
Localization of Nbp1p at the SPB and its genetic interactions with NDC1 and MPS2 prompted us to investigate whether Nbp1p physically associates with Ndc1p and Mps2p. Initially, we conducted two-hybrid assays and found interactions between Nbp1p and Ndc1p, and Nbp1p and Mps2p (Figure 5A). Interestingly, Nbp1p binding was compromised when the mutated ndc1-39 two-hybrid construct was used (Figure 5A). The Nbp1p-Ndc1p interaction is therefore likely to be important for SPB duplication. In addition, Ndc1p did not bind to Mps2p (Figure 5A), suggesting Nbp1p may mediate the interaction between Ndc1p and Mps2p as revealed by genetics. Importantly, Nbp1p showed specificity for a subset of membrane proteins at the SPB, as we did not observe Nbp1p binding to Kar1p (Figure 5A) or to Mps3p (unpublished observation). Finally, Nbp1p failed to interact with Bbp1p (Figure 5A), which is known to bind to Mps2p (Schramm et al., 2000). Thus, the interaction between Nbp1p and Mps2p may be direct and not mediated by Bbp1p. In conclusion, these results suggest that Nbp1p physically interacts with Ndc1p and Mps2p.
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). Nbp1p-TAP was bound to IgG beads and then eluted by Tev protease as described in Materials and Methods. Using this condition, we could show coimmunoprecipitation between Nbp1p-TAP and Bbp1p-6HA (Figure 5B). Extracts from the control NBP1 MPS2-6HA strain revealed a small amount of Mps2p-6HA in the Tev eluate (Figure 5B). However, we repeatedly recovered 2-3 times more Mps2p-6HA when extracts from NBP1-TAP MPS2-6HA cells were used (Figure 5B), consistent with the two-hybrid result. Therefore, we conclude that Nbp1p interacts with the Bbp1p-Mps2p complex.
Cells Lacking Nbp1p Fail in SPB Duplication and Exhibit a Mitotic Cell Cycle Arrest
NBP1's genetic interactions and the finding that Nbp1p is in complex with Mps2p and Bbp1p suggests that Nbp1p is required for SPB duplication, which would be revealed by the phenotypes of conditional alleles of the gene. However, several attempts to obtain a temperature-sensitive nbp1(ts) allele failed. We therefore constructed temperature-sensitive NBP1-degron (nbp1-td) cells. In these cells, NBP1 was fused to the degron element, which targets proteins for degradation at 37°C (Dohmen et al., 1994). To ensure efficient degradation, UBR1, encoding a ubiquitin-protein ligase, was expressed from the inducible GAL1 promoter (Kanemaki et al., 2003). In the presence of Ubr1p almost all of the Nbp1p-td protein became degraded when cells were shifted to 37°C (Figure 6A). Moreover, analysis of nbp1-td-GFP cells by fluorescence microscopy revealed that upon shifting cells to 37°C, the Nbp1p-td-GFP signal was no longer detected and therefore did not colocalize with the Spc42-eqFP-marked SPBs (Figure 6B). This data suggests that most of the cellular Nbp1p-td is targeted for degradation at 37°C.
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To examine the effects of Nbp1p depletion, nbp1-td cells were first synchronized in G1-phase by the addition of -factor. Cells were then washed to remove -factor and shifted to 37°C in the presence of UBR1 induction to trigger Nbp1p-td degradation. Analysis of the DNA content by flow cytometry (unpublished observations) and of the budding index indicated that nbp1-td cells replicated the DNA as wild-type cells but then were arrested in the first cell cycle with a 2C DNA content and a large bud (Figure 6D). We went onto to ask whether nbp1-td cells have a defect in spindle formation. Microtubules were visualized by expression of CFP--tubulin (CFP-TUB1). In addition, SPC72-YFP was expressed in order to mark the outer plaque of the SPB, which is assembled after the duplication plaque is inserted into the nuclear envelope. Most nbp1-td CFP-TUB1 SPC72-YFP cells (>95%) failed to assemble a bipolar spindle (Figure 6, C and D). About 60% of these cells arrested with a single Spc72p-YFP signal, indicating a defect in SPB duplication or separation. The remainder 
40% of cells showed two Spc72p-YFP signals of equal intensity. In these cells, the SPB in the mother cell body carried a thick bundle of nuclear microtubules, whereas the SPB in the bud was only associated with 1-2 microtubules. All microtubules of the bud SPB were directed toward the cell cortex and probably represent cytoplasmic microtubules. The daughter SPB is therefore devoid of most nuclear microtubules. Consistently, the nuclear DNA was still within the mother cell. Thus, Nbp1p has an essential function during the SPB duplication process.
Nbp1p Is Required to Insert the Duplication Plaque into the Nuclear Envelope
Electron microscopy was performed in order to better understand the SPB defect of nbp1-td cells. SPB substructures, such as the bridge, satellite, and the half-bridge can be visualized this way (Byers and Goetsch, 1975). Synchronized wild-type and nbp1-td cells were prepared for thin-section electron microscopy after 1 h at 37°C. All analyzed wild-type cells had fully duplicated the SPB and formed a short metaphaselike bipolar spindle. In contrast, nbp1-td cells showed the mother SPB still associated via the bridge structure with the extended duplication plaque (Figure 7B). The duplication plaque was not embedded into the nuclear envelope, and it did not contain nuclear microtubules, which were only associated with the mother SPB. Strikingly, in some of the nbp1-td cells, the duplication plaque was found on the cytoplasmic side of the nuclear envelope in the daughter cell away from the mother SPB (Figure 7C). The observed "dead" pole phenotype is reminiscent of conditional lethal ndc1, bbp1, and mps2 cells. These gene products have an essential function in insertion of the duplication plaque into the nuclear envelope (Winey et al., 1991, 1993; Munoz-Centeno et al., 1999; Schramm et al., 2000). Thus, it appears that Nbp1p is also required for insertion of the duplication plaque into the nuclear envelope, most likely as a component of the Mps2p-Bbp1p complex and in cooperation with Ndc1p.
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). We reasoned that proteins interacting with or functioning downstream of Nbp1p may not be associated with the defective SPB in nbp1-td cells. We first analyzed synchronized nbp1-td SPC42-eqFP cells either expressing BBP1-GFP or MPS2-GFP. At 135 min after the release from the -factor block, 96% of large-budded wild-type cells were in anaphase and showed colocalization of Spc42p-eqFP and Bbp1p-GFP (Figure 8A). In contrast, 61% of nbp1-td cells were large-budded and contained two Spc42-eqFP signals. Of these cells, about half of them did not have Bbp1p-GFP associated with the defective new SPB located in the bud (Figure 8A). However, Bbp1p-GFP was still associated with the mother SPB. This suggests that in a high proportion of nbp1-td cells, Bbp1p fails to associate with the newly formed SPB, whereas it remains associated with the mother SPB. The localization dependency was even more pronounced in the case of Mps2p. In nbp1-td cells, Mps2p-GFP was neither associated with the mother nor the daughter SPB (Figure 8B). Furthermore, Mps2-GFP is degraded upon inactivation of Nbp1-td (unpublished observation). This result is consistent with the notion that Mps2p binds to SPBs through Nbp1p and that Mps2p is not stable when not at the SPB.
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; Elliott et al., 1999).
Nbp1p Binds with Reduced Efficiency to the New SPB of mps2-1 Cells
Binding of Mps2p to the SPB was most dramatically affected in nbp1-td cells (Figure 8), raising the possibility that Mps2p binds to SPBs via Nbp1p. Inactivation of Mps2p may therefore also affect SPB association of Nbp1p. To test this possibility, we examined the localization of Nbp1p-GFP in mps2-1, mps2-2, or bbp1-1 cells, which are mutated in the genes encoding components of the Mps2p-Bbp1p complex. Analysis of synchronized wild-type, mps2-1, mps2-2, and bbp1-1 cells showed that SPB localization of Nbp1p-GFP was only affected in mps2-1 cells (Figure 9). Of the 53% of large-budded mps2-1 cells with two Spc42-eqFP signals, about half of them carried only one Nbp1p-GFP signal. In most of these cells, the defective new SPB in the bud was devoid of an Nbp1p-GFP signal. Thus, SPB localization of Nbp1p is in part dependent on Mps2p, reinforcing the importance of the interaction of Nbp1p and Mps2p in SPB duplication.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), even though Nbp1p was not identified in a previous mass spectrometry study aimed at revealing SPB components (Wigge et al., 1998). However, the mass spectrometry study did not identify several peripheral SPB components including Mps3p, Sfi1p, Kar1p, and Ndc1p, possibly because of the nature of the SPB preparation and/or abundance of these components. Nonetheless, we show here that Nbp1p is a new part of the Mps2p-Bbp1p complex (Schramm et al., 2000) and that Nbp1p is required to insert the nascent SPB into the nuclear envelope, similar to Ndc1p, Mps2p, and Bbp1p (Winey et al., 1991, 1993; Schramm et al., 2000).
Besides SPB duplication defects, ndc1-39 mutants also fail in NPC assembly (Lau et al., 2004). It is likely that extra copies of NBP1 suppress only the SPB duplication defects in ndc1-39 mutants, because Nbp1p only localizes to the SPBs and not to the NPCs. The assertion that Nbp1p is at the SPB is based on fluorescence microscopy and immunoelectron microscopy with tagged alleles of the gene. However, both of these approaches have detection limits making it impossible to rule out other cellular localizations for Nbp1p. Furthermore, the very useful nbp1-td allele may not reveal other non-SPB-related functions of NBP1 because of the first cell cycle arrest of cells depleted for Nbp1p. The identification of other Nbp1p functions, if any, is likely to require new alleles and/or the identification and analysis of additional binding partners.
NBP1-like Genes
Using a BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), we identified clear Nbp1p homologues in other budding yeast species (see Supplementary Figure S1) but not in fission yeast or higher eukaryotes, which is also true for Bbp1p. In addition, the protein encoded by S. cerevisiae YPR174C displays significant homology to the C-terminal half (residues 188-304) of Nbp1p (35% identical, 52% similar). Like Nbp1p, the protein encoded by YPR174C also contains a coiled-coil domain and has been localized to the SPB and nuclear periphery by fluorescence microscopy (Huh et al., 2003; Sundin et al., 2004). Both promoters of NBP1 and YPR174C contain a MluI "cell cycle box" and in both cases the mRNA expression peaks during G1 (Spellman et al., 1998; Shimizu et al., 2000), consistent with playing a role during SPB duplication. Furthermore, both Nbp1p and Ypr174p contain cyclin-dependent kinase consensus phosphorylation sites and they have been shown to function as in vitro substrates of the cyclin-dependent kinase Cdc28p (Ubersax et al., 2003). Interestingly, Nbp1p becomes phosphorylated in mid-S-phase (H. Maekawa, unpublished observations), consistent with the finding that Cdc28p-Clb5p may perform this phosphorylation. However, unlike NBP1, YPR174C is not essential and cell cycle analysis of ypr174c cells did not show a SPB duplication defect (H. Maekawa, unpublished observations). Thus, the YPR174C gene product cannot be required for SPB duplication. Although NBP1 is involved in SPB duplication, the cellular function of YPR174C remains unknown.
Nbp1p Functions in SPB Duplication
As mentioned above, we used the nbp1-td allele to show that cells depleted of Nbp1p fail in SPB duplication and come to a mitotic arrest as is common for SPB mutants (reviewed in Jaspersen and Winey, 2004). Importantly, the terminal phenotype observed in these cells indicated that an aberrant, or "dead" SPB had been assembled, and this SPB morphology was confirmed by electron microscopy. Strains mutant in MPS2, NDC1, or BBP1 exhibit a similar defect in SPB duplication (Winey et al., 1991, 1993; Schramm et al., 2000), which has been interpreted as a defect in the insertion of the nascent SPB into the nuclear envelope. Our finding that Nbp1p is in a complex with Mps2p and Bbp1p and interacts with Ndc1p is consistent with the common phenotype of nbp1-td and conditional lethal bbp1, mps2, and ndc1 cells.
The failure to insert the nascent SPB results in the formation of a "dead" SPB that resides on the cytoplasmic face of the nuclear envelope. It is not thought to be a normal intermediate in SPB duplication, which can be assembled to the point of forming cytoplasmic microtubules (Winey et al., 1991, 1993; Schramm et al., 2000). To have this function, the outer plaque must be assembled, and the failure to form microtubules in the nucleus arises from the fact that "dead" SPB does not have access to the nucleus and lacks an inner plaque as revealed by electron microscopy (Winey et al., 1991, 1993; Schramm et al., 2000). The "dead" SPB formed in nbp1-td cells migrates into the new cell like the "dead" pole in other mutants that form this structure (Jaspersen and Winey, 2004), whereas the new SPB generally stays in the mother cell in wild-type cells (Pereira et al., 2001). This could arise from unusual mobility of the "dead" SPB because it not restrained by nuclear microtubules. Furthermore, the inner plaque component, Spc110p is absent from "dead" SPBs in bbp1-1 mutants (Schramm et al., 2000). We extend that analysis here to show that the "dead" SPBs in nbp1-td cells contain the central plaque components Spc29p and Spc42p, but lack the inner plaque component Spc110p, as expected. The "dead" SPBs have reduced levels of Nbp1p's binding partners, Mps2p and Bbp1p. Interestingly, the bridge components Cdc31p, Kar1p, Mps3p, and Sfi1p are all present at the "dead" SPB. The reduced levels of the Mps2p-Bbp1p complex may be sufficient to retain the half-bridge, and/or Ndc1p may mediate the interaction. Also, the fact that half-bridge is associated with the "dead" SPB at all may be a remnant of the attachment of the duplication plaque to the half-bridge such that the half-bridge is broken as the cytoplasmic microtubules pull on the "dead" SPB. Alternatively, the bridge may go through its normal separation process that requires mitotic CDK activity in the absence of SPB insertion. It would be intriguing if normal bridge severing occurred, because such a finding would indicate that some steps in the SPB cycle do not depend on completion of earlier steps. Furthermore, it would be interesting to analyze the composition of the "dead" SPBs in the different mutants that form these aberrant structures.
Although the precise mechanism of SPB insertion into the nuclear envelope is unknown, Adams and Kilmartin (1999) suggested fusion of the inner and outer nuclear membranes at the SPB-distal end of the bridge. We propose that during SPB duplication, Nbp1p interacts with the Mps2p-Bbp1p complex and likely with Ndc1p to promote fusion of the inner and outer nuclear membranes for the insertion of the duplication plaque into the nuclear envelope.
Beyond functioning in SPB insertion, Ndc1p, Mps2p, Bbp1p, and Nbp1p all remain at the SPB after assembly. It has been proposed that the Mps2p-Bbp1p complex serves as a tether to hold the SPB in place, where Mps2p is the membrane-bound component of the complex and Bbp1p is the bridge to the SPB via interaction with Spc29p (Schramm et al., 2000). It is possible that Nbp1p has a similar bridging function. Beyond being part of the Mps2p-Bbp1p complex, NBP1 displayed a genetic interaction with SPC42, and both genetic and two-hybrid interactions with NDC1. These interactions suggest that Nbp1p may bridge between the membrane proteins, Mps2p and Ndc1p, and/or bridge from the membrane proteins to Spc42p in the central plaque of the SPB (Figure 10). It is tempting to speculate that a complex including Nbp1p and Bbp1p form the "hooklike" structure between the central plaque and the nuclear envelope observed by high-voltage electron tomography (O'Toole et al., 1999; Schramm et al., 2000).
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Finally, NBP1 is somewhat unusual among genes encoding SPB components in that it acts as a dosage suppressor of mutations in a variety of genes encoding SPB components required of SPB duplication. Furthermore, although Nbp1p is clearly part of the Mps2p-Bbp1p complex, the immunoelectron microscopy data suggests that it could be at other SPB substructures. Taken together, these results suggest Nbp1p could have a very central role in SPB duplication, possibly through interactions with multiple SPB components. New alleles of NBP1 and/or the identification and analysis of additional binding partners in the SPB will be required in order to reveal other non-SPB functions and additional SPB functions of Nbp1p.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: CFP, cyan fluorescent protein; DAPI, 4',6-diamidono-2-phenylindole; GFP, green fluorescent protein; NPC, nuclear pore complex; ORF, open reading frame; SPB, spindle pole body; ts, temperature-sensitive.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Present address: Zentrum, ZMBH, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany;
|| Present address: Section of Cell and Developmental Biology, Division of Biological Sciences, 0347, University of California, San Diego, La Jolla, CA 92093-0347;
¶ Present address: Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110.
Address correspondence to: Mark Winey (Mark.Winey{at}colorado.edu).
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