Stefan Irniger

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.

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 modules 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.

Two different modes of cyclin Clb2 proteolysis during mitosis in Saccharomyces cerevisiae

Sister chromatid separation and mitotic exit are triggered by the anaphase‐promoting complex (APC/C) which is a multi‐subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate‐specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1‐dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1‐independent mechanism.

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.

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.

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.

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 APCCdh1 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 APCCdh1 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 APCCdh1.

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 Alzheimers disease. In our experimental setting loss of APC2 form principle forebrain neurons did not affect the course of pathology in an Alzheimers disease mouse model. In conclusion, our data provides genetic evidence that APC/C activity in the adult forebrain is required for cognitive function.

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

ABSTRACT Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G1 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 SCFCdc4 ubiquitin ligase and requires phosphorylation by the G1 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 G1 Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

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.