Rey-Huei Chen

Mps1 Phosphorylation by MAP Kinase Is Required for Kinetochore Localization of Spindle-Checkpoint Proteins

The spindle checkpoint delays anaphase onset until all chromosomes have achieved bipolar attachment to the spindle microtubules. Unattached kinetochores activate the spindle checkpoint by recruiting several spindle-checkpoint proteins, including Mps1, Mad1, Mad2, Bub1, Bub3, and BubR1 (Mad3 in yeast). In vertebrate cells, active MAP kinase (MAPK) is also enriched at unattached kinetochores and is required for the spindle checkpoint. It has been shown that the kinase activity of Mps1 is required for the spindle checkpoint and for kinetochore localization of Bub1, Bub3, Mad1, and Mad2 . We herein demonstrate that MAPK phosphorylates Mps1 at S844 in Xenopus egg extracts. Interestingly, changing S844 to unphosphorylatable alanine (S844A) has no effect on the kinase activity of Mps1, although it abolishes the checkpoint function of Mps1. Biochemical and immunofluorescence studies show that S844A mutation perturbs kinetochore localization of Mps1 and other spindle-checkpoint proteins, whereas the phosphorylation-mimicking S844D mutant restores their functions. Our studies suggest that Mps1 phosphorylation by MAPK at S844 might create a phosphoepitope that allows Mps1 to interact with kinetochores. In addition, our results indicate that active Mps1 must localize to kinetochores in order to execute its checkpoint function.

Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

The metazoan nuclear envelope breaks down in early mitosis and does not reform until late anaphase. Phosphorylation of lamin B receptor by Cdk1 not only prevents premature nuclear envelope assembly, but also facilitates complete dissociation of the nuclear envelope from the chromatin during nuclear envelope breakdown.

The AAA-ATPase Cdc48 and cofactor Shp1 promote chromosome bi-orientation by balancing Aurora B activity

The assembly, disassembly and dynamic movement of macromolecules are integral to cell physiology. The ubiquitin-selective chaperone Cdc48 (p97 in Metazoa), an AAA-ATPase, might facilitate such processes in the cell cycle. Cdc48 in budding yeast was initially isolated from a mitotic mutant. However, its function in mitosis remained elusive. Here we show that the temperature-sensitive cdc48-3 mutant and depletion of cofactor Shp1 (p47 in Metazoa) cause cell-cycle arrest at metaphase. The arrest is due to a defect in bipolar attachment of the kinetochore that activates the spindle checkpoint. Furthermore, Cdc48-Shp1 positively regulates Glc7/protein phosphatase 1 by facilitating nuclear localization of Glc7, whereas it opposes Ipl1/Aurora B kinase activity. Thus, we propose that Cdc48-Shp1 promotes nuclear accumulation of Glc7 to counteract Ipl1 activity. Our results identify Cdc48 and Shp1 as critical components that balance the kinase and phosphatase activities at the kinetochore in order to achieve stable bipolar attachment.

Cdc48 and cofactors Npl4-Ufd1 are important for G1 progression during heat stress by maintaining cell wall integrity in Saccharomyces cerevisiae.

The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities) ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD), ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.

Lipid droplets maintain lipid homeostasis during anaphase for efficient cell separation in budding yeast

Excess lipids are stored in the form of neutral lipids in lipid droplets. The inability to convert excess lipids into neutral lipids during anaphase creates a lipid imbalance that perturbs the normal dynamics of cytokinesis molecules, causing a delay in cell separation.

Assembly and quality control of the protein phosphatase 1 holoenzyme involves the Cdc48–Shp1 chaperone

ABSTRACT Protein phosphatase 1 (PP1) controls many aspects of cell physiology, which depends on its correct targeting in the cell. Nuclear localization of Glc7, the catalytic subunit of PP1 in budding yeast, requires the AAA-ATPase Cdc48 and its adaptor Shp1 through an unknown mechanism. Herein, we show that mutations in SHP1 cause misfolding of Glc7 that co-aggregates with Hsp104 and Hsp42 chaperones and requires the proteasome for clearance. Mutation or depletion of the PP1 regulatory subunits Sds22 and Ypi1, which are involved in nuclear targeting of Glc7, also produce Glc7 aggregates, indicating that association with regulatory subunits stabilizes Glc7 conformation. Use of a substrate-trap Cdc48QQ mutant reveals that Glc7–Sds22–Ypi1 transiently associates with and is the major target of Cdc48–Shp1. Furthermore, Cdc48–Shp1 binds and prevents misfolding of PP1-like phosphatases Ppz2 and Ppq1, but not other types of phosphatases. Our data suggest that Cdc48–Shp1 functions as a molecular chaperone for the structural integrity of PP1 complex in general and that it specifically promotes the assembly of Glc7–Sds22–Ypi1 for nuclear import.

Lipid droplets are central organelles for meiosis II progression during yeast sporulation

During meiosis II, lipids are remodeled and incorporated into a double-membrane structure termed the prospore membrane (PSM), which grows as it sequesters four haploid nuclei to form spores. Cellular organelle lipid droplets are recruited to the PSM, where they play an active role in organizing membrane morphogenesis.

Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect

BackgroundIn mammalian cells, ASPL is involved in insulin-stimulated redistribution of the glucose transporter GLUT4 and assembly of the Golgi apparatus. Its putative yeast orthologue, Ubx4, is important for proteasome localization, endoplasmic reticulum-associated protein degradation (ERAD), and UV-induced degradation of RNA polymerase.ResultsHere, we show that ASPL is a cofactor of the hexameric ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast. In addition, ASPL interacts in vitro with NSF, another hexameric ATPase complex. ASPL localizes to the ER membrane. The central area in ASPL, containing both a SHP box and a UBX domain, is required for binding to the p97 N-domain. Knock-down of ASPL does not impair degradation of misfolded secretory proteins via the ERAD pathway. Deletion of UBX4 in yeast causes cycloheximide sensitivity, while ubx4 cdc48-3 double mutations cause proteasome mislocalization. ASPL alleviates these defects, but not the impaired ERAD.ConclusionsIn conclusion, ASPL and Ubx4 are homologous proteins with only partially overlapping functions. Both interact with p97/Cdc48, but while Ubx4 is important for ERAD, ASPL appears not to share this function.

Cdc48 Chaperone and Adaptor Ubx4 Distribute the Proteasome in the Nucleus for Anaphase Proteolysis

Background: Anaphase progression requires efficient degradation of mitotic regulators. Results: Mutations in the chaperone Cdc48-Ubx4 perturb anaphase proteolysis and proteasome distribution. Conclusion: Cdc48-Ubx4 maintains an optimal level of proteasomes for anaphase proteolysis. Significance: The molecular chaperone Cdc48-Ubx4 links proteasome mobilization with mitosis. The cell cycle transition is driven by abrupt degradation of key regulators. While ubiquitylation of these proteins has been extensively studied, the requirement for the proteolytic step is less understood. By analyzing the cell cycle function of Cdc48 in the budding yeast Saccharomyces cerevisiae, we found that double mutations in Cdc48 and its adaptor Ubx4 cause mitotic arrest with sustained Clb2 and Cdc20 proteins that are normally degraded in anaphase. The phenotype is neither caused by spindle checkpoint activation nor a defect in the assembly or the activity of the ubiquitylation machinery and the proteasome. Interestingly, the 26S proteasome is mislocalized into foci, which are colocalized with nuclear envelope anchor Sts1 in cdc48-3 ubx4 cells. Moreover, genetic analysis reveals that ubx4 deletion mutant dies in the absence of Rpn4, a transcriptional activator for proteasome subunits, and the proteasome chaperone Ump1, indicating that an optimal level of the proteasome is required for survival. Overexpression of Rpn4 indeed can rescue cell growth and anaphase proteolysis in cdc48-3 ubx4 cells. Biochemical analysis further shows that Ubx4 interacts with the proteasome. Our data propose that Cdc48-Ubx4 acts on the proteasome and uses the chaperone activity to promote its nuclear distribution, thereby optimizing the proteasome level for efficient degradation of mitotic regulators.