The lipid droplet assembly complex consists of seipin and four accessory factors in budding yeast.

Seipin, a crucial protein for cellular lipid droplet (LD) assembly, oligomerizes at the interface between the endoplasmic reticulum (ER) and LDs to facilitate neutral lipid packaging. Using proximity labeling, we identify four proteins-Ldo45, Ldo16, Tgl4, and Pln1-that are recruited to the vicinity of yeast seipin, the Sei1-Ldb16 complex, exclusively when seipin function is intact, hence termed seipin accessory factors. Localization studies identify Tgl4 at the ER-LD contact site, in contrast to Ldo45, Ldo16 and Pln1 at the LD surface. Cells with compromised seipin function resulted in uneven distribution of these proteins with aberrant LDs, supporting a central role of seipin in orchestrating their association with the LD. Overexpression of any seipin accessory factor causes LD aggregation and affects a subset of LD protein distribution, highlighting the importance of their stoichiometry. Although single factor mutations show minor LD morphology changes, combined mutations have additive effects. Lastly, we present evidence that seipin accessory factors assemble and interact with seipin in the absence of neutral lipids and undergo dynamically rearrangements during LD formation induction, with Ldo45 acting as a central hub recruiting other factors to interact with the seipin complex.

Lipid saturation induces degradation of squalene epoxidase for sterol homeostasis and cell survival.

A fluid membrane containing a mix of unsaturated and saturated lipids is essential for life. However, it is unclear how lipid saturation might affect lipid homeostasis, membrane-associated proteins, and membrane organelles. Here, we generate temperature-sensitive mutants of the sole fatty acid desaturase gene OLE1 in the budding yeast Saccharomyces cerevisiae Using these mutants, we show that lipid saturation triggers the endoplasmic reticulum-associated degradation (ERAD) of squalene epoxidase Erg1, a rate-limiting enzyme in sterol biosynthesis, via the E3 ligase Doa10-Ubc7 complex. We identify the P469L mutation that abolishes the lipid saturation-induced ERAD of Erg1. Overexpressed WT or stable Erg1 mutants all mislocalize into foci in the ole1 mutant, whereas the stable Erg1 causes aberrant ER and severely compromises the growth of ole1, which are recapitulated by doa10 deletion. The toxicity of the stable Erg1 and doa10 deletion is due to the accumulation of lanosterol and misfolded proteins in ole1 Our study identifies Erg1 as a novel lipid saturation-regulated ERAD target, manifesting a close link between lipid homeostasis and proteostasis that maintains sterol homeostasis under the lipid saturation condition for cell survival.

Chromosome detachment from the nuclear envelope is required for genomic stability in closed mitosis.

Mitosis in metazoans involves detachment of chromosomes from the nuclear envelope (NE) and NE breakdown, whereas yeasts maintain the nuclear structure throughout mitosis. It remains unknown how chromosome attachment to the NE might affect chromosome movement in yeast. By using a rapamycin-induced dimerization system to tether a specific locus of the chromosome to the NE, I found that the tethering delays the separation and causes missegregation of the region distal to the tethered site. The phenotypes are exacerbated by mutations in kinetochore components and Aurora B kinase Ipl1. The chromosome region proximal to the centromere is less affected by the tether, but it exhibits excessive oscillation before segregation. Furthermore, the tether impacts full extension of the mitotic spindle, causing abrupt shrinkage or bending of the spindle in shortened anaphase. The study supports detachment of chromosomes from the NE being required for faithful chromosome segregation in yeast and segregation of tethered chromosomes being dependent on a fully functional mitotic apparatus.

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

Neutral lipids, predominantly triacylglycerol (TAG) and sterol ester, are stored within the cellular organelles termed lipid droplets (LDs). Although it is believed that the major function of LDs is to supply the cell with energy and membranes, little is known about the cellular events directly involving LDs and their contents. In this study, we provide cytological evidence that LDs form direct contacts with the prospore membrane (PSM) that is synthesized de novo during meiosis II to sequester the dividing nuclei in sporulating yeast. Lipidomic analyses indicate that TAG lipolysis releases free fatty acids at a time that correlates well with meiosis II progression, concomitant with phospholipid remodeling. Mutants lacking TAG or impaired of TAG hydrolysis show spore wall assembly defects, supporting a role for TAG and/or its metabolites in spore wall morphogenesis. Not only does LD integrity influence spore wall assembly, LDs are also essential for other aspects of spore development. Yeast cells lacking LDs are severely defective in PSM growth and organization and display disrupted spindles, producing dead spores or even failing to form spores. Together these results link LD physiology directly to a unique membrane morphogenesis process critical for development.

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

The neutral lipids steryl ester and triacylglycerol (TAG) are stored in the membrane-bound organelle lipid droplet (LD) in essentially all eukaryotic cells. It is unclear what physiological conditions require the mobilization or storage of these lipids. Here, we study the budding yeast mutant are1Δ are2Δ dga1Δ lro1Δ, which cannot synthesize the neutral lipids and therefore lacks LDs. This quadruple mutant is delayed at cell separation upon release from mitotic arrest. The cells have abnormal septa, unstable septin assembly during cytokinesis, and prolonged exocytosis at the division site at the end of cytokinesis. Lipidomic analysis shows a marked increase of diacylglycerol (DAG) and phosphatidic acid, the precursors for TAG, in the mutant during mitotic exit. The cytokinesis and separation defects are rescued by adding phospholipid precursors or inhibiting fatty acid synthesis, which both reduce DAG levels. Our results suggest that converting excess lipids to neutral lipids for storage during mitotic exit is important for proper execution of cytokinesis and efficient cell separation.

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

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 Cdc48(QQ) 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.

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

BACKGROUND: In 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. RESULTS: Here, 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. CONCLUSIONS: In 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.

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.

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

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

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.

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.

The spindle checkpoint in Xenopus laevis.

The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase onset until all kinetochores have properly established bipolar attachment to spindle microtubules. This mechanism is important for all eukaryotic cells and is evolutionarily conserved. Much of our understanding of the molecular and biochemical mechanisms of the spindle checkpoint has been gained from parallel studies in various experimental systems. In particular, the cytoplasmic extract from the eggs of Xenopus laevis provides an unsurpassable system for biochemical analysis of the spindle checkpoint and has made important contributions to the field. This article reviews the progress of the spindle checkpoint studies in Xenopus laevis with a focus on the regulation by phosphorylation.

Dual inhibition of Cdc20 by the spindle checkpoint.

The metaphase-to-anaphase transition is triggered by the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that targets proteins for degradation, leading to sister chromatid separation and mitotic exit. The function of APC is controlled by the spindle checkpoint that delays anaphase onset in the presence of any chromosome that has not established bipolar attachment to the mitotic spindle. In this way, the checkpoint ensures accurate chromosome segregation. The spindle checkpoint is mostly activated from kinetochores that are not attached to microtubules or not under tension that is normally generated from bipolar attachment. These kinetochores recruit several spindle checkpoint proteins to assemble an inhibitory complex composed of checkpoint proteins Mad2, Bub3, and Mad3/BubR1. This complex binds and inhibits Cdc20, an activator and substrate adaptor for APC. In addition, the checkpoint complex promotes Cdc20 degradation, thus lowering Cdc20 protein level upon checkpoint activation. This dual inhibition on Cdc20 likely ensures that the spindle checkpoint is sustained even when the cell contains only a single unattached kinetochore.

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.

Phosphorylation and activation of Bub1 on unattached chromosomes facilitate the spindle checkpoint.

The spindle checkpoint inhibits anaphase until all kinetochores have attached properly to spindle microtubules. The protein kinase Bub1 is an essential checkpoint component that resides at kinetochores during mitosis. It is shown herein that Xenopus Bub1 becomes hyperphosphorylated and the kinase is activated on unattached chromosomes. MAP kinase (MAPK) contributes to this phosphorylation, as inhibiting MAPK or altering MAPK consensus sites in Bub1 to alanine or valine (Bub1(5AV)) abolishes the phosphorylation and activation on chromosomes. Both Bub1 and Bub1(5AV) support the checkpoint under an optimal condition for spindle checkpoint activation. However, Bub1, but not Bub1(5AV), supports the checkpoint at a relatively low concentration of nuclei or the microtubule inhibitor nocodazole. Similar to Bub1(5AV), Bub1 without the kinase domain (Bub1(deltaKD)) is also partially compromised in its checkpoint function and in its ability to recruit other checkpoint proteins to kinetochores. This study suggests that activation of Bub1 at kinetochores enhances the efficiency of the spindle checkpoint and is probably important in maintaining the checkpoint toward late prometaphase when the cell contains only a few or a single unattached kinetochore.

Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae.

The spindle checkpoint arrests cells at the metaphase-to-anaphase transition until all chromosomes have properly attached to the mitotic spindle. Checkpoint proteins Mad2p and Mad3p/BubR1p bind and inhibit Cdc20p, an activator for the anaphase-promoting complex (APC). We find that upon spindle checkpoint activation by microtubule inhibitors benomyl or nocodazole, wild-type Saccharomyces cerevisiae contains less Cdc20p than spindle checkpoint mutants do, whereas their CDC20 mRNA levels are similar. The difference in Cdc20p levels correlates with their difference in the half-lives of Cdc20p, indicating that the spindle checkpoint destabilizes Cdc20p. This process requires the association between Cdc20p and Mad2p, and functional APC, but is independent of the known destruction boxes in Cdc20p and the other APC activator Cdh1p. Importantly, destabilization of Cdc20p is important for the spindle checkpoint, because a modest overexpression of Cdc20p causes benomyl sensitivity and premature Pds1p degradation in cells treated with nocodazole. Our study suggests that the spindle checkpoint reduces Cdc20p to below a certain threshold level to ensure a complete inhibition of Cdc20p before anaphase.

Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint.

The spindle checkpoint delays anaphase until all chromosomes are properly attached to spindle microtubules. When the spindle checkpoint is activated at unattached kinetochores, the checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC). Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68. Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X. laevis egg extracts. However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the spindle checkpoint signal, owing to their reduced affinity for the spindle checkpoint proteins. This study demonstrates that the spindle checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20. Our results also have implications for the spindle checkpoint silencing mechanism.

BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1.

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.

Molecular interpretation of ERK signal duration by immediate early gene products.

The duration of intracellular signalling is associated with distinct biological responses, but how cells interpret differences in signal duration are unknown. We show that the immediate early gene product c-Fos functions as a sensor for ERK1 (extracellular-signal-regulated kinase 1) and ERK2 signal duration. When ERK activation is transient, its activity declines before the c-Fos protein accumulates, and under these conditions c-Fos is unstable. However, when ERK signalling is sustained, c-Fos is phosphorylated by still-active ERK and RSK (90K-ribosomal S6 kinase). Carboxy-terminal phosphorylation stabilizes c-Fos and primes additional phosphorylation by exposing a docking site for ERK, termed the FXFP (DEF) domain. Mutating the DEF domain disrupts the c-Fos sensor and c-Fos-mediated signalling. Other immediate early gene products that control cell cycle progression, neuronal differentiation and circadium rhythms also contain putative DEF domains, indicating that multiple sensors exist for sustained ERK signalling. Together, our data identify a general mechanism by which cells can interpret differences in ERK activation kinetics.

Spindle checkpoint requires Mad1-bound and Mad1-free Mad2.

The spindle checkpoint prevents anaphase from occurring until all chromosomes have attached properly to the mitotic spindle. The checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the checkpoint. We now demonstrate that in Xenopus egg extracts Mad1 and Mad2 form a stable complex, whereas a fraction of Mad2 molecules is not bound to Mad1. The checkpoint establishment and maintenance are lost upon titrating out free Mad2 with an excess of Mad1 or a truncated Mad1 (amino acids 326-718, Mad1C) that contains the Mad2-binding region. Mad1N (amino acids 1-445) that binds kinetochores, but not Mad2, reduces Mad1 and Mad2 at kinetochores and abolishes checkpoint maintenance. Furthermore, the association between Mad2 and Cdc20, the activator for the anaphase-promoting complex, is enhanced under checkpoint-active condition compared with that at metaphase. Immunodepletion analysis shows that the Mad1-free Mad2 protein is unable to bind Cdc20, consistent with the model that kinetochore localization of Mad2 facilitates the formation of Mad2-Cdc20 complex. This study demonstrates that the ratio between Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that is necessary for the spindle checkpoint. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.