Functional Characterization of Dma1 and Dma2, the Budding Yeast Homologues ofSchizosaccharomyces pombeDma1 and Human Chfr

Abstract

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1⌬ dma2⌬ cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase. INTRODUCTIONFaithful chromosome segregation requires the mitotic spindle to be correctly positioned with respect to the cell division axis. Cytokinesis must take place only after this task is achieved and the two sets of chromosomes are sufficiently far apart. Unscheduled cytokinesis before proper chromosome segregation would, in fact, lead to formation of anucleate and polyploid cells. Eukaryotic cells prevent these harmful events by a surveillance mechanism, called spindle position checkpoint, that delays cytokinesis in the presence of mispositioned or misoriented spindles until errors are corrected. This control is particularly critical for organisms, such yeasts and higher plants, that specify the site of cytokinesis before spindle assembly.In the budding yeast Saccharomyces cerevisiae, the mitotic spindle first gets positioned close to the bud neck, the constriction where cytokinesis will take place later on, and then crosses the neck to enter the bud, dragging the nucleus along. Spindle positioning and orientation involve a searchand-capture process and occur in two steps, which depend on two partially overlapping pathways (Schuyler and Pellman, 2001). During the first step cytoplasmic microtubules (cMTs) try to find and interact with specific sites on the bud neck cortex. The EB1/Bim1 protein, localized at the plus end of cMTs, is directly involved in searching cortical sites by promoting cMTs dynamicity (Tirnauer et al., 1999). Nuclear positioning also requires the cortical protein Kar9, with which Bim1 interacts (M...

Combining chemical genetics and proteomics to identify protein kinase substrates

“…Among the five bud neck and bud localized substrates are three of the six Pcl1-specific substrates, including a structural component of the septin ring and known regulators of septin dynamics and actin polarization. An additional target, Dma1, plays a role in septin ring assembly (33). Colocalization of these substrates and Pcl1-Pho85 further suggests that the substrates we have identified are specific for this kinase.…”

Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes

“…These included Hsl1 and Gin4, which are important for sensing septin organization and initiating the morphogenesis checkpoint (52). We also identified proteins involved in mitotic spindle orientation, Dma2, a protein with a role in spindle positioning (53), and Dyn1, a protein that is partially functionally redundant with Kar9 (36). Lte1, a component of the mitotic exit network, was also identified as a Cdc14-D253A interactor.…”

Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

“…Multiple isolates of SPC24 and its interacting partner SPC25 were recovered (Janke et al, 2001;Wigge and Kilmartin, 2001). We also identified four genes encoding proteins that regulate spindle dynamics-MT-associated proteins Stu1 and Stu2, the kinesinrelated motor protein Kip2, and a protein involved in spindle positioning called Dma1 (Roof et al, 1992;Pasqualone and Huffaker, 1994;Wang and Huffaker, 1997;Fraschini et al, 2004). Two protein kinases were isolated-Mck1, which has a role in chromosome segregation, and Rck2, which has a role in the osmotic stress response pathway (Neigeborn and Mitchell, 1991;Shero and Hieter, 1991;Bilsland-Marchesan et al, 2000).…”