The FEAR Protein Slk19 Restricts Cdc14 Phosphatase to the Nucleus until the End of Anaphase, Regulating Its Participation in Mitotic Exit in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of SLK19, slk193R (three lysine-to-arginine mutations), strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to slk19Δ cells, slk193R cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike slk19Δcells, however, slk193R cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, slk193R cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: slk193R causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that slk193R partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.

Exploring Novel Functions of the Small GTPase Ypt1p under Heat-Shock by Characterizing a Temperature-Sensitive Mutant Yeast Strain, ypt1-G80D

“…This was similar for the recombinant proteins analyzed on native PAGE (Figure 3B, upper gel). Considering that Glycine, the 80th residue of Ypt1p is replaced with Aspartic acid in Ypt1p G80D , the amino acid replacement could affect size shifts on the gel by affecting secondary or tertiary structure of the protein [13,14].…”

Slk19 enhances cross-linking of microtubules by Ase1 and Stu1

“…For anaphase, the nature of this role is at least partially defined (Sullivan et al, 2001;Stegmeier et al, 2002;Khmelinskii et al, 2007;Havens et al, 2010;Faust et al, 2013), for metaphase it is not. To start with, it is unclear whether Slk19 localizes to the spindle MTs in metaphase, in contrast to anaphase, when Slk19 clearly localizes to the spindle midzone.…”

“…A MT minus end directed motor (Kar3/Cik1) facilitates the alignment of antiparallel ipMTs between the spindle poles either per se (Hepperla et al, 2014) or in association with the MT plus end tracking protein Bim1 (Kornakov et al, 2020) and plus-end directed motors (Cin8 and Kip1) provide a force that slides the ipMTs apart (Saunders and Hoyt, 1992). Stu1, a protein that belongs to the CLASP family of MT rescue factors, is essential for the stabilization of the nuclear spindle MTs (Yin et al, 2002) and also may contribute to the crosslinking of ipMTs up to metaphase (Funk et al, 2014). In addition, Ase1, as its orthologs in other eucaryotes, crosslinks ipMTs in metaphase and at the spindle midzone in anaphase (Schuyler et al, 2003;Bieling et al, 2010;Subramanian et al, 2010).…”

Estudio de la función y regulación de la proteína Cdc14A en el ciclo de división celular

“…ScCdc14 es una proteína esencial para la salida de mitosis, en la que inhibe a los complejos CDK mitóticos y desfosforila varios de sus sustratos (Bardin and Amon, 2001;Jaspersen et al, 1999;Visintin et al, 1998). El orden de los procesos que tienen lugar en esta fase del ciclo viene determinado por la regulación en varios pasos de la localización y activación de ScCdc14 gracias a las rutas FEAR (Fourteen Early Anaphase Release, liberación de ScCdc14 en anafase temprana) y MEN (Mitotis Exit Network, ruta de salida de mitosis) (Azzam et al, 2004;Bardin et al, 2000;Faust et al, 2013;Jaspersen et al, 1998;Jensen et al, 2002;Palani et al, 2012;Shou et al, 1999;Stegmeier et al, 2002). ScCdc14 se encuentra localizada en el nucléolo durante interfase y el inicio de mitosis por la unión a su inhibidor Net1.…”

“…ScCdc14 se encuentra localizada en el nucléolo durante interfase y el inicio de mitosis por la unión a su inhibidor Net1. Al inicio de anafase, gracias a la acción de la ruta FEAR, comienza a ser liberada de esta localización y pasa al núcleo, al huso mitótico y al SPB (Spindle Pole Body, cuerpo polar del huso, análogo del centrosoma en células humanas) (Azzam et al, 2004;Faust et al, 2013;Stegmeier et al, 2002).…”