Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.     

酿酒酵母Sfi1p的结构及生物学功能

纺锤体极体(spindle pole body,SPB)是酵母细胞的微管组织中心,它在细胞分裂及细胞遗传稳定性的维持过程中起着极其重要的作用,是细胞生物学领域热门的研究方向.Sfi1p是酿酒酵母SPB的必需蛋白并且横跨整个半桥,该蛋白与SPB的复制有关,它的缺失或突变会导致SPB复制失败,在哺乳动物的中心体也存在酵母Sfi1p的同源蛋白.本文系统的介绍了酵母Sfi1p及其在人类中心体中的同源蛋白hSfi1p的结构特征,并且阐明了Sfi1p在SPB复制与分离、核配及生孢等细胞周期过程中的作用.对Sfi1p的功能研究,将有助于解决SPB研究过程中重要的科学问题,同时为中心体中Sfi1p同源蛋白的功能研究提供良好的借鉴.     

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.     

Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.     

Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.     

Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.     

Localization of core spindle pole body (SPB) components during SPB duplication in Saccharomyces cerevisiae.

We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.     

The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G₁ the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G₁. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.     

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.     

Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1

Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

Lessons from yeast: the spindle pole body and the centrosome

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was obtained from a reconstituted complex, the γ-tubulin complex, and also from a sub-particle, SPB cores, prepared by extraction of an enriched SPB preparation. The labelling of SPB proteins with GFP at the N or C termini, using GFP tags inserted into the genome, gave informative electron microscopy localization and fluorescence resonance energy transfer data. Examples are given of more precise functional data obtained by removing domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the differences between SPB and centrosome duplication discussed.     

Yeast ubiquitin-like genes are involved in duplication of the microtubule organizing center

KAR1 is required for duplication of the Saccharomyces cerevisiae microtubule organizing center, the spindle pole body (SPB) (Rose, M.D., and G.R. Fink, 1987. Cell. 48:1047-1060). Suppressors of a kar1 allele defective for SPB duplication were isolated in two genes, CDC31 and DSK2 (Vallen, E.A., W.H., M. Winey, and M.D. Rose. 1994. Genetics. 137:407-422). To elucidate the role of DSK2 in SPB duplication, we cloned the gene and found it encodes a novel ubiquitin-like protein containing an NH2 terminus 36% identical to ubiquitin. The only other known yeast ubiquitin-like protein is encoded by the nucleotide excision repair gene RAD23 (Watkins, J.F.,P. Sung, L. Prakash, and S. Prakash. 1993. Mol. Cell. Bio. 13:7757-7765). Unlike ubiquitin, the NH2- terminal domain of Dsk2p is not cleaved from the protein, indicating that Dsk2p is not conjugated to other proteins. Although the DSK2-1 mutation alters a conserved residue in the Dsk2p ubiquitin-like domain, we detect no differences in Dsk2p or Cdc31p stability. Therefore, DSK2 does not act by interfering with ubiquitin-dependent protein degradation of these proteins. Although DSK2 is not essential, a strain deleted for both DSK2 and RAD23 is temperature sensitive for growth due to a block in SPB duplication. In addition, overexpression of DSK2 is toxic, and the DSK2-1 allele causes a block in SPB duplication. Therefore, DSK2 dosage is critical for SPB duplication. We determined that CDC31 gene function is downstream of DSK2 and KAR1. Dsk2p is a nuclear-enriched protein, and we propose that Dsk2p assists in Cdc31 assembly into the new SPB.     

Asymmetric mitotic segregation of the yeast spindle pole body.

The yeast KAR1 gene is required for spindle pole body (SPB) duplication and nuclear fusion. We determine here that KAR1-beta-galactosidase hybrid proteins localize to the outer face of the SPB. Remarkably, after SPB duplication, the hybrid protein was found associated with only one of the two SPBs, usually the one that enters the bud. Using an ndc1 mutant, which forms a defective SPB at the nonpermissive temperature, we found that the hybrid was exclusively associated with the "new" SPB. Two regions of KAR1 contribute to its localization; an internal 70 residue region was necessary and sufficient to localize hybrids to the SPB, and the hydrophobic carboxyl terminus localized proteins to the nuclear envelope. The localization domains correspond to two functional domains required for SPB duplication. We suggest that KAR1 is anchored to the nuclear envelope and interacts with at least one other SPB component during the cell cycle.     

Novel sfi1 alleles uncover additional functions for Sfi1p in bipolar spindle assembly and function

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 microm apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle assembly.     

Modulation of L-type Ca2+ channels by gbeta gamma and calmodulin via interactions with N and C termini of alpha 1C

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).     

Terminally Repeated Sequences on a Herpesvirus Genome Are Deleted following Circularization but Are Reconstituted by Duplication during Cleavage and Packaging of Concatemeric DNA

The mechanisms underlying cleavage of herpesvirus genomes from replicative concatemers are unknown. Evidence from herpes simplex virus type 1 suggests that cleavage occurs by a nonduplicative process; however, additional evidence suggests that terminal repeats may also be duplicated during the cleavage process. This issue has been difficult to resolve due to the variable numbers of reiterated terminal repeats that the herpes simplex virus type 1 genome can contain. Guinea pig cytomegalovirus is a herpesvirus with a simple terminal repeat arrangement that defines two genome types. Type II genomes have a single copy of a 1-kb terminal repeat at both their left and right termini, whereas type I genomes have only one copy at their left termini and lack the repeat at their right termini. In a previous study, we constructed a recombinant guinea pig cytomegalovirus in which certain cis elements were disrupted such that only type II genomes were produced. Here we show that double repeats that are formed by circularization of infecting genomes are rapidly converted to single repeats, such that the junctions between genomes within replicative concatemers formed late in infection almost exclusively contain single copies of the terminal repeat. Therefore, for the recombinant virus, each cleavage event begins with a single repeat within a concatemer yet produces two repeats, one at each of the resulting termini, demonstrating that terminal repeat duplication occurs in conjunction with cleavage. For wild-type guinea pig cytomegalovirus, the formation of type I genomes further suggests that cleavage can also occur by a nonduplicative process and that duplicative and nonduplicative cleavage can occur concurrently. Other herpesviruses having terminal repeats, such as the herpes simplex viruses and human cytomegalovirus, may also utilize repeat duplication and deletion; however, the biological importance of these events remains unknown.     

Tropomyosin requires an intact N-terminal coiled coil to interact with tropomodulin

Tropomodulins (Tmods) are tropomyosin (TM) binding proteins that bind to the pointed end of actin filaments and modulate thin filament dynamics. They bind to the N termini of both "long" TMs (with the N terminus encoded by exon 1a of the alpha-TM gene) and "short" nonmuscle TMs (with the N terminus encoded by exon 1b). In this present study, circular dichroism was used to study the interaction of two designed chimeric proteins, AcTM1aZip and AcTM1bZip, containing the N terminus of a long or a short TM, respectively, with protein fragments containing residues 1 to 130 of erythrocyte or skeletal muscle Tmod. The binding of either TMZip causes similar conformational changes in both Tmod fragments promoting increases in both alpha-helix and beta-structure, although they differ in binding affinity. The circular dichroism changes in the Tmod upon binding and modeling of the Tmod sequences suggest that the interface between TM and Tmod includes a three- or four-stranded coiled coil. An intact coiled coil at the N terminus of the TMs is essential for Tmod binding, as modifications that disrupt the N-terminal helix, such as removal of the N-terminal acetyl group from AcTM1aZip or striated muscle alpha-TM, or introduction of a mutation that causes nemaline myopathy, Met-8-Arg, into AcTM1aZip destroyed Tmod binding.     

Position dependence of the 13C chemical shifts of alpha-helical model peptides. Fingerprint of the 20 naturally occurring amino acids

The position dependence of the (13)C chemical shifts was investigated at the density functional level for alpha-helical model peptides represented by the sequence Ac-(Ala)(i)-X-(Ala)(j)-NH(2), where X represents any of the 20 naturally occurring amino acids, with 0 < or = i < or = 8 and i + j = 8. Adoption of the locally dense basis approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 20 naturally occurring amino acids in alpha-helices, there is (1) significant variability of the computed (13)C shielding as a function of both the guest residue (X) and the position along the sequence; for example, at the N terminus, the (13)C(alpha) and (13)C(beta) shieldings exhibit a uniform pattern of variation with respect to both the central or the C-terminal positions; (2) good agreement between computed and observed (13)C(alpha) and (13)C(beta) chemical shifts in the interior of the helix, with correlation coefficients of 0.98 and 0.99, respectively; for (13)C(alpha) chemical shifts, computed in the middle of the helix, only five residues, namely Asn, Asp, Ser, Thr, and Leu, exhibit chemical shifts beyond the observed standard deviation; and (3) better agreement for four of these residues (Asn, Asp, Ser, and Thr) only for the computed values of the (13)C(alpha) chemical shifts at the N terminus. The results indicate that (13)C(beta), but not (13)C(beta), chemical shifts are sensitive enough to reflect the propensities of some amino acids for specific positions within an alpha-helix, relative to the N and C termini of peptides and proteins.     

Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.