Elmar Schiebel

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR‐primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C‐terminal tagged genes or for the exchange of the promoter and N‐terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C‐terminal epitope tags as well as nine different promoter substitutions in combination with N‐terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast‐optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches. Copyright

Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines

Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR‐based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope‐tagged proteins. Copyright

Evidence that the Ipl1-Sli15 (Aurora Kinase-INCENP) Complex Promotes Chromosome Bi-orientation by Altering Kinetochore-Spindle Pole Connections

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin.

The Bub2p spindle checkpoint links nuclear migration with mitotic exit.

Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.

A novel protein complex promoting formation of functional alpha- and gamma-tubulin.

We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4‐1, encoding a mutated yeast γ‐tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co‐immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4‐1 and super‐sensitive towards the microtubule‐depolymerizing drug benomyl. All except Δgim4 are cold‐sensitive and their microtubules disassemble at 14°C. The Gim proteins have one function related to α‐tubulin and another to Tub4p, supported by the finding that the benomyl super‐sensitivity is caused by a reduced level of α‐tubulin while the synthetic lethality with tub4‐1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4‐1 as well as the cold‐sensitivity and benomyl super‐sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional α‐ and γ‐tubulin.

Modes of spindle pole body inheritance and segregation of the Bfa1p–Bub2p checkpoint protein complex

Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre‐existing ‘old’ and a newly formed SPB. The two SPBs of yeast cells are functionally distinct. It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p–Bub2p GTPase‐activating protein (GAP) complex, a component of the spindle positioning checkpoint. We investigated whether the functional difference of the two SPBs correlates with the time of their assembly. We describe that in unperturbed cells the ‘old’ SPB always migrates into the bud. However, Bfa1p localization is not determined by SPB inheritance. It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p–Bub2p GAP to the bud‐ward‐localized SPB. In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p–Bub2p complex binds to both SPBs. This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle. Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p–Bub2p GAP accordingly.

Spc98p and Spc97p of the yeast γ‐tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p

Previously, we have shown that the yeast γ‐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.

Compartmentation of protein folding in vivo: sequestration of non‐native polypeptide by the chaperonin–GimC system

The functional coupling of protein synthesis and chaperone‐assisted folding in vivo has remained largely unexplored. Here we have analysed the chaperonin‐dependent folding pathway of actin in yeast. Remarkably, overexpression of a heterologous chaperonin which traps non‐native polypeptides does not interfere with protein folding in the cytosol, indicating a high‐level organization of folding reactions. Newly synthesized actin avoids the chaperonin trap and is effectively channelled from the ribosome to the endogenous chaperonin TRiC. Efficient actin folding on TRiC is critically dependent on the hetero‐oligomeric co‐chaperone GimC. By interacting with folding intermediates and with TRiC, GimC accelerates actin folding at least 5‐fold and prevents the premature release of non‐native protein from TRiC. We propose that TRiC and GimC form an integrated ‘folding compartment’ which functions in cooperation with the translation machinery. This compartment sequesters newly synthesized actin and other aggregation‐sensitive polypeptides from the crowded macromolecular environment of the cytosol, thereby allowing their efficient folding.

The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control

Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24‐2 and spc25‐7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24‐2 and spc25‐7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24‐2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

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

Previously, we have shown that the γ‐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.