The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism.

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK module's proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope.

Aggregate clearance of α-synuclein in Saccharomyces cerevisiae depends more on autophagosome and vacuole function than on the proteasome.

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.

The Ime2 protein kinase family in fungi: more duties than just meiosis.

Ime2 of the budding yeast Saccharomyces cerevisiae belongs to a family of conserved protein kinases displaying sequence similarities to both cyclin-dependent kinases and mitogen-activated protein kinases. Ime2 has a pivotal role for meiosis and sporulation. The involvement of this protein kinase in the regulation of various key events in meiosis, such as the initiation of DNA replication, the expression of meiosis-specific genes and the passage through the two consecutive rounds of nuclear divisions has been characterized in detail. More than 20 years after the identification of the IME2 gene, a recent report has provided the first evidence for a function of this gene outside of meiosis, which is the regulation of pseudohyphal growth. In the last few years, Ime2-related protein kinases from various fungal species were studied. Remarkably, these homologues are not generally required for meiosis, but instead have other specific tasks. In filamentous ascomycete species, Ime2 homologues are involved in the inhibition of fruiting body formation in response to environmental signals. In the pathogenic basidiomycetes Ustilago maydis and Cryptococcus neoformans, members of this kinase family apparently have primary roles in regulating mating. Thus, Ime2-related kinases exhibit an amazing variety in controlling sexual developmental programs in fungi.

The anaphase promoting complex is required for memory function in mice.

Learning and memory processes critically involve the orchestrated regulation of de novo protein synthesis. On the other hand it has become clear that regulated protein degradation also plays a major role in neuronal plasticity and learning behavior. One of the key pathways mediating protein degradation is proteosomal protein destruction. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets proteins for proteosomal degradation by the 26S proteasome. While the APC/C is essential for cell cycle progression it is also expressed in postmitotic neurons where it has been implicated with axonal outgrowth and neuronal survival. In this study we addressed the role of APC/C in learning and memory function by generating mice that lack the essential subunit APC2 from excitatory neurons of the adult forebrain. Those animals are viable but exhibit a severe impairment in the ability to extinct fear memories, a process critical for the treatment of anxiety diseases such as phobia or post-traumatic stress disorder. Since deregulated protein degradation and APC/C activity has been implicated with neurodegeneration we also analyzed the effect of Apc2 deletion in a mouse model for Alzheimer's disease. In our experimental setting loss of APC2 form principle forebrain neurons did not affect the course of pathology in an Alzheimer's disease mouse model. In conclusion, our data provides genetic evidence that APC/C activity in the adult forebrain is required for cognitive function.

LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity.

VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.

Fungal development and the COP9 signalosome.

The conserved COP9 signalosome (CSN) multiprotein complex is located at the interface between cellular signaling, protein modification, life span and the development of multicellular organisms. CSN is required for light-controlled responses in filamentous fungi. This includes the circadian rhythm of Neurospora crassa or the repression of sexual development by light in Aspergillus nidulans. In contrast to plants and animals, CSN is not essential for fungal viability. Therefore fungi are suitable models to study CSN composition, activity and cellular functions and its role in light controlled development.

Dissection of mitotic functions of the yeast cyclin Clb2.

Progression through mitosis requires the activity of cyclin-dependent kinases (CDKs) associated with regulatory cyclin subunits. In the yeast Saccharomyces cerevisiae, Clb2 has the most important role among the four mitotic cyclins, Clb1-4, manifested by data showing that simultaneous deletion of the CLB1, CLB3 and CLB4 genes has only minor effects on mitosis. Thus, Clb2 alone is sufficient for all essential CDK functions in mitosis, such as the assembly of bipolar spindles and spindle elongation. Here, we show that a modification of Clb2, by the C-terminal addition of a Myc12 epitope, causes the loss of one specific mitotic function of Clb2. Strains carrying CLB2-MYC12 are nonviable in the absence of the CLB3 and CLB4 genes, because the modified Clb2 version fails to promote assembly of the mitotic spindle. In contrast, Clb2-Myc12 has no apparent defects in late mitotic functions and, furthermore, induces the switch from polarized to isotropic growth with similar efficiency as the endogenous Clb2. Thus, the presence of the Myc12 epitope selectively inactivates Clb2's capacity to promote spindle formation. Clb2-Myc12 represents therefore the first version of Clb2 impaired in one specific mitotic function. We conclude that the major mitotic functions of this cyclin can be unequivocally dissected.

The protein kinase ImeB is required for light-mediated inhibition of sexual development and for mycotoxin production in Aspergillus nidulans.

Spore formation is a common process in the developmental cycle of fungi. In the yeast Saccharomyces cerevisiae, Ime2 is a key protein kinase for the meiotic cell cycle, which precedes ascospore formation. Here, we analysed the IME2-related imeB gene of the filamentous ascomycete Aspergillus nidulans. imeB deletion strains are retarded in growth and overproduce fertile sexual fruiting bodies in the presence of light, which normally represses sexual development. imeB mutants also display abnormal differentiation of sexual Hülle cells in submerged cultures. Increased sexual development of imeB mutants is dependent on VeA, a component of the heterotrimeric velvet complex. A combined deletion of imeB with the phytochrome fphA, a red light receptor, results in a complete loss of light response, suggesting that ImeB and FphA cooperate in light-mediated inhibition of sexual development. Furthermore, we found that imeB mutants fail to produce the mycotoxin sterigmatocystin, an aflatoxin precursor, and show that ImeB is needed for expression of the sterigmatocystin gene cluster. ImeB contains a TXY motif conserved in mitogen-activated protein kinases. This sequence element is essential for ImeB function. We conclude that ImeB is a mitogen-activated protein kinase-related protein kinase required for the co-ordinated control of light-dependent development with mycotoxin production.

Nucleus-specific and cell cycle-regulated degradation of mitogen-activated protein kinase scaffold protein Ste5 contributes to the control of signaling competence.

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G(1) phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF(Cdc4) ubiquitin ligase and requires phosphorylation by the G(1) cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G(1) Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

The C-terminal region of the meiosis-specific protein kinase Ime2 mediates protein instability and is required for normal spore formation in budding yeast.

The cyclin-dependent kinase Cdk1 and the related kinase Ime2 act in concert to trigger progression of the meiotic cell cycle in the yeast Saccharomyces cerevisiae. These kinases share several functions and substrates during meiosis, but their regulation seems to be clearly different. In contrast to Cdk1, no cyclin seems to be involved in the regulation of Ime2 activity. Ime2 is a highly unstable protein, and we aimed to elucidate the relevance of Ime2 instability. We first determined the sequence elements required for Ime2 instability by constructing a set of deletions in the IME2 gene. None of the small deletions in Ime2 affected its instability, but deletion of a 241 amino acid C-terminal region resulted in a highly stabilized protein. Thus, the C-terminal domain of Ime2 is important for mediating protein instability. The stabilized, truncated Ime2 protein is highly active in vivo. Replacement of the IME2 gene with the truncated IME2DeltaC241 in diploid strains did not interfere with meiotic nuclear divisions, but caused abnormalities in spore formation, as manifested by the appearance of many asci with a reduced spore number such as triads and dyads. The truncated Ime2 caused a reduction of spore number in a dominant manner. We conclude that downregulation of Ime2 kinase activity mediated by the C-terminal domain is required for the efficient production of normal four-spore asci. Our data suggest a role for Ime2 in spore number control in S. cerevisiae.

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5.

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.

Preventing fatal destruction: inhibitors of the anaphase-promoting complex in meiosis.

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin-ligase whose major functions in the cell cycle are the initiation of sister chromatid separation and the inactivation of cyclin-dependent kinases. This complex is also essential for meiosis, a specialized form of the cell cycle characterized by two consecutive rounds of chromosome segregation. To ensure a proper meiotic cell cycle, the activity of APC/C needs to be tightly controlled. It is now evident that inhibitors of APC/C play pivotal roles to avert its untimely activation. During prophase I, this ubiquitin-ligase must be kept inactive to prevent precocious sister chromatid separation. Studies in yeast showed that this inhibition is mediated by a specific subunit of the complex. Accurate chromosome segregation in meiosis I depends on spindle checkpoint proteins such as Mad2 which delay APC/C activation in response to an erroneous spindle attachment of chromosomes. Additional APC/C antagonists are known to block complete cyclin destruction between meiosis I and II, thereby ensuring that cyclin dependent kinases remain active and that DNA replication does not occur. Inhibitors of APC/C also mediate the cytostatic factor induced metaphase II arrest of oocytes. This review highlights the current knowledge about the role and relevance of these diverse regulators of the meiotic APC/C.

Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast.

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.

Controlling transcription by destruction: the regulation of yeast Gcn4p stability.

The Gcn4 protein, a member of the AP-1 family of transcription factors, is involved in the expression of more than 500 genes in the budding yeast Saccharomyces cerevisiae. A key role of Gcn4p is the increased expression of many amino acid biosynthesis genes in response to amino acid starvation. The accumulation of this transcription activator is mainly induced by efficient translation of the GCN4 ORF and by stabilisation of the Gcn4 protein. Under normal growth conditions, Gcn4p is a highly unstable protein, thereby resembling many eukaryotic transcription factors, including mammalian Jun and Myc proteins. Gcn4p is degraded by ubiquitin-dependent proteolysis mediated by the Skp1/cullin/F-box (SCF) ubiquitin ligase, which recognises specifically phosphorylated substrates. Two cyclin-dependent protein kinases, Pho85p and Srb10p, have crucial functions in regulating Gcn4p phosphorylation and degradation. The past few years have revealed many novel insights into these regulatory processes. Here, we summarise current knowledge about the factors and mechanisms regulating Gcn4p stability.

Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1-3 proteins in Saccharomyces cerevisiae.

Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis. APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases. The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins. This study shows that glucose and the activated Ras2(Val19) protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast. Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways. Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium. Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function. However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3. Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway.

Cyclin destruction in mitosis: a crucial task of Cdc20.

Proteolytic destruction of cyclins is a fundamental process for cell division. At the end of mitosis, degradation of mitotic cyclins results in the inactivation of cyclin-dependent kinases. Cyclin proteolysis is triggered by the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit complex which contains ubiquitin ligase activity. Recent data in yeast demonstrated that a partial degradation of the mitotic cyclin Clb2, mediated by APC/C and its activator protein Cdc20, is essential and sufficient for the mitotic exit. Remarkably, a complete inactivation of cyclin-dependent kinases seems to be not essential. This review discusses recent novel insights into cyclin destruction and its implications for the mitotic exit.

Amino acid-dependent Gcn4p stability regulation occurs exclusively in the yeast nucleus.

The c-Jun-like transcriptional activator Gcn4p controls biosynthesis of translational precursors in the yeast Saccharomyces cerevisiae. Protein stability is dependent on amino acid limitation and cis signals within Gcn4p which are recognized by cyclin-dependent protein kinases, including Pho85p. The Gcn4p population within unstarved yeast consists of a small relatively stable cytoplasmic fraction and a larger less stable nuclear fraction. Gcn4p contains two nuclear localization signals (NLS) which function independently of the presence or absence of amino acids. Expression of NLS-truncated Gcn4p results in an increased cytoplasmic fraction and an overall stabilization of the protein. The same effect is achieved for the entire Gcn4p in a yrb1 yeast mutant strain impaired in the nuclear import machinery. In the presence of amino acids, controlled destabilization of Gcn4p is triggered by the phosphorylation activity of Pho85p. A pho85delta mutation stabilizes Gcn4p without affecting nuclear import. Pho85p is localized within the nucleus in the presence or absence of amino acids. Therefore, there is a strict spatial separation of protein synthesis and degradation of Gcn4p in yeast. Control of protein stabilization which antagonizes Gcn4p function is restricted to the nucleus.

Inhibition of APC-mediated proteolysis by the meiosis-specific protein kinase Ime2.

Proteolysis triggered by the anaphase-promoting complex (APC) is needed for sister chromatid separation and the exit from mitosis. APC is a ubiquitin ligase whose activity is tightly controlled during the cell cycle. To identify factors involved in the regulation of APC-mediated proteolysis, a Saccharomyces cerevisiae GAL-cDNA library was screened for genes whose overexpression prevented degradation of an APC target protein, the mitotic cyclin Clb2. Genes encoding G1, S, and mitotic cyclins were identified, consistent with previous data showing that the cyclin-dependent kinase Cdk1 associated with different cyclins is a key factor for inhibiting APC(Cdh1) activity from late-G1 phase until mitosis. In addition, the meiosis-specific protein kinase Ime2 was identified as a negative regulator of APC-mediated proteolysis. Ectopic expression of IME2 in G1 arrested cells inhibited the degradation of mitotic cyclins and of other APC substrates. IME2 expression resulted in the phosphorylation of Cdh1 in G1 cells, indicating that Ime2 and Cdk1 regulate APC(Cdh1) in a similar manner. The expression of IME2 in cycling cells inhibited bud formation and caused cells to arrest in mitosis. We show further that Ime2 itself is an unstable protein whose proteolysis occurs independently of the APC and SCF (Skp1/Cdc53/F-box) ubiquitin ligases. Our findings suggest that Ime2 represents an unstable, meiosis-specific regulator of APC(Cdh1).