Gislene Pereira

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.

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.

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.

The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment.

Tub4p is a novel tubulin found in Saccharomyces cerevisiae. It most resembles gamma‐tubulin and, like it, is localized to the yeast microtubule organizing centre, the spindle pole body (SPB). In this paper we report the identification of SPC98 as a dosage‐dependent suppressor of the conditional lethal tub4–1 allele. SPC98 encodes an SPB component of 98 kDa which is identical to the previously described 90 kDa SPB protein. Strong overexpression of SPC98 is toxic, causing cells to arrest with a large bud, defective microtubule structures, undivided nucleus and replicated DNA. The toxicity of SPC98 overexpression was relieved by co‐overexpression of TUB4. Further evidence for an interaction between Tub4p and Spc98p came from the synthetic toxicity of tub4–1 and spc98–1 alleles, the dosage‐dependent suppression of spc98–4 by TUB4, the binding of Tub4p to Spc98p in the two‐hybrid system and the co‐immunoprecipitation of Tub4p and Spc98p. In addition, Spc98–1p is defective in its interaction with Tub4p in the two‐hybrid system. We suggest a model in which Tub4p and Spc98p form a complex involved in microtubule organization by the SPB.

Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery.

Centrins are calmodulin-like proteins that function in the duplication of microtubule-organizing centres. Here we describe a new function of the yeast centrin Cdc31. We show that overproduction of a sequence, termed CID, in the carboxy-terminal domain of the nuclear export factor Sac3 titrates Cdc31, causing a dominant-lethal phenotype and a block in spindle pole body (SPB) duplication. Under normal conditions, the CID motif recruits Cdc31 and Sus1 (a subunit of the SAGA transcription complex) to the Sac3–Thp1 complex, which functions in mRNA export together with specific nucleoporins at the nuclear basket. A previously reported cdc31 temperature-sensitive allele, which is neither defective in SPB duplication nor Kic1 kinase activation, induces mRNA export defects. Thus, Cdc31 has an unexpected link to the mRNA export machinery.

Regulation of the Bfa1p–Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p–Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p–Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.

Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis

Cep164 provides a molecular link between the mother centriole and the ciliary membrane biogenesis machinery by interacting with the GEF Rabin8 and the GTPase Rab8.

Tandem fluorescent protein timers for in vivo analysis of protein dynamics

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.