Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels and localization

Abstract

Swe1 is a key regulator of mitosis, and its levels are tightly regulated in response to different stress conditions. Budding yeast Dma1 and Dma2 contribute to the control of Swe1 localization, ubiquitylation, and degradation.

Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis

“…Accordingly, the isolation of Swe1 mutant variants that are more stable but do not show a defect in their interaction with any of the known Swe1 regulators (9) indicates the existence of other proteins that control Swe1 activity and abundance. In accordance with other studies (7,12), we previously reported that Swe1 is not completely degraded after mitotic entry during an unperturbed cell cycle (11), and in this paper we confirm that Swe1 is present in metaphase-arrested cells, consistent with a physiological role for Swe1 after mitotic entry. In addition, here we show that Swe1 is phosphorylated and mostly localized in the nucleus and in the cytoplasm of the mother cell during metaphase, and then, during anaphase and concomitantly with nuclear division, it is dephosphorylated and spread also into the bud.…”

“…Swe1-3HA localization was observed in formaldehyde-fixed cells as described in Ref. 11. Digital images were taken with a Leica DC350F charge-coupled device camera mounted on a Nikon Eclipse 600 and controlled by the Leica FW4000 software or with the MetaMorph imaging system software on a fluorescent microscope (Eclipse 90i; Nikon), equipped with a charge-coupled device camera (Coolsnap, Photometrics) with an oil 100ϫ 0.5-1.3 PlanFluor oil objective (Nikon).…”

“…During the S phase, Swe1 accumulates in the nucleus where it is phosphorylated by Clb-Cdc28 before being exported to the cytoplasm and then to the daughter side of the bud neck. Bud neck-localized Swe1 is phosphorylated by several protein kinases (10), and hyperphosphorylated Swe1 species are ubiquitylated (9,11). Subsequently, Swe1 is degraded via the proteasome, and this event allows mitotic entry (9).…”

“…Subsequently, Swe1 is degraded via the proteasome, and this event allows mitotic entry (9). Little is known about the role of Swe1 after this point of the cell cycle, although it is reported that a pool of Swe1 persists in the cells after mitotic entry (11,12), suggesting that it might also play a role in mitotic progression.…”

Vhs2 is a novel regulator of septin dynamics in budding yeast

“…Phosphatase assay was performed as previously described. 49 For western blot analysis, proteins were transferred to Protran membranes (Schleicher and Schuell) and probed with monoclonal anti-HA (1:3000), anti-Pgk1 antibodies (1:40 000), and anti-FLAG antibodies (1:3000). Secondary antibodies were purchased from Amersham, and proteins were detected by an enhanced chemioluminescence system according to the manufacturer.…”