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 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 SCF^Cdc4 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.Scaffold proteins play a pivotal role in spatial and temporal regulation of multitiered mitogen-activated protein kinase (MAPK) cascades (8, 30, 107). Scaffold protein function can be controlled at several different levels, including phosphorylation, oligomerization, and subcellular localization, which can dramatically influence signaling (5, 21, 61).A well-characterized scaffold-dependent MAPK pathway drives the mating pheromone response in budding yeast Saccharomyces cerevisiae (15). The occupancy of a heterotrimeric G-protein-coupled receptor by pheromone results in release of its associated membrane-tethered Gβγ (Ste4-Ste18) complex. Ste5 scaffold protein (917 residues) is recruited to the plasma membrane via its association with this freed Gβγ (106) and by additional multivalent contacts with membrane phospholipids mediated by an N-terminal amphipathic α-helix (PM motif) (111) and an internal PH domain (34). Because Ste5 is also able to bind a MAPK kinase kinase (Ste11), a MAPK kinase (Ste7), and two MAPKs (Fus3 and Kss1) (102), membrane recruitment of Ste5 delivers these components to the plasma membrane. Membrane localization of Ste5 juxtaposes its passenger kinases to Ste20, a p21-activated protein kinase that also interacts with membrane phospholipids (94) and requires plasma membrane-tethered and GTP-loaded Cdc42 for its activation (56, 58, 60). GTP-bound Cdc42 is generated in this vicinity via other Gβγ-recruited effectors, especially Far1, which binds the Cdc42 guanine nucleotide exchange factor, Cdc24 (14, 98). Once activated, Ste20 directly phosphorylates and activates the Ste11 MAPK kinase kinase, triggering the MAPK cascade (24, 114).In naïve haploid cells, Ste5 undergoes continuous nucleocytoplasmic shuttling but is located predominantly in the nucleus (53, 66). In response to pheromone, this flux is dramatically shifted in favor of export, elevating the cytosolic pool of Ste5, thereby raising the number of molecules available for membrane recruitment (66, 79). Pheromone-induced nuclear export of Ste5 requires the exportin, Msn5/Ste21 (66).Little is known about why Ste5 is located in the nucleus in unstimulated cells. It has been suggested that passage of Ste5 through the nucleus modifies it in an as yet undefined manner to make it “competent” to subsequently promote signaling at the membrane (66, 103). However, other evidence indicates that nuclear shuttling of Ste5 is not necessary for its translocation to the plasma membrane or its function (34, 79, 111) and that reimport into the nucleus contributes to pathway downregulation following initial stimulation (53). It has remained obscure, mechanistically speaking, how nuclear localization of Ste5 contributes to the regulation of pathway activation and signal flux.Given that Ste5 is the least abundant component of this entire signaling system (≤500 molecules per haploid cell) (38), we suspected that dynamic regulation of the location and level of this scaffold protein provides a critically important control point for influencing the timing, potency, duration, and specificity of signaling in this pathway. Indeed, as described here, we found that the subcellular localization of Ste5 and cell cycle progression have dramatic effects in controlling the stability of Ste5. Our findings provide new insights about the physiological importance of Ste5 nuclear localization and G₁ cyclin-dependent protein kinase 1 (CDK1) action in establishment and maintenance of the conditions that preserve signaling fidelity in this system.     

Genomic Plasticity of the Human Fungal Pathogen Candida albicans

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.The purpose of this review is to describe the tools used to detect genome changes, to highlight recent advances in our understanding of large-scale chromosome changes that arise in Candida albicans, and to discuss the role of specific stresses in eliciting these genome changes. The types of genomic diversity that have been characterized suggest that C. albicans can undergo extreme genomic changes in order to survive stresses in the human host. We propose that C. albicans and other pathogens may have evolved mechanisms not only to tolerate but also to generate large-scale genetic variation as a means of adaptation.C. albicans is a polymorphic yeast with a 16-Mb (haploid) genome organized in 8 diploid chromosomes (140, 154, 203). The C. albicans genome displays a very high degree of plasticity. This plasticity includes the types of genomic changes frequently observed with cancer cells, including gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity (reviewed in references 100, 117, and 157). Similar to somatic cancer cells, C. albicans reproduces primarily through asexual clonal division (65, 84). Nonetheless, it has retained much of the machinery needed for mating and meiosis (189), yet meiosis has never been observed (13, 120).C. albicans has two mating-type-like (MTL) alleles, MTLa and MTLα (76). The MTL locus is on the left arm of chromosome 5 (Chr5), approximately 80 kbp from the centromere. Most C. albicans isolates are heterozygous for the MTL locus, but approximately 3 to 10% of clinical isolates are naturally homozygous at MTL (104, 108). Mating can occur between strains carrying the opposite MTL locus, and most strains that were found to be naturally MTL homozygous are mating competent (104, 108). MTL-homozygous strains were also constructed from MTL-heterozygous strains by deletion of either the MTLa or MTLα locus (77) or by selection for Chr5 loss on sorbose (87, 115).Mating between these diploid strains of opposite mating type can occur both in vitro (115) and in vivo (77, 97). The products are tetraploid and do not undergo a conventional meiotic reduction in ploidy (12, 120). Rather, they undergo random loss of multiple chromosomes, a process termed “concerted chromosome loss,” until they reach a near-diploid genome content (2, 12, 53, 85). A subset of these cells also undergoes multiple gene conversion events reminiscent of meiotic recombination, and most remain trisomic for one to several chromosomes (53). While mating and concerted chromosome loss have been induced in the laboratory, the role of the parasexual cycle during the host-pathogen interaction and in the response to stresses, such as exposure to antifungal drugs, remains unclear. The prevailing model is that adaptive mutations (such as those that occur with the acquisition of drug resistance) evolve through somatic events, including point mutations, recombination, gene conversion, loss of heterozygosity, and/or aneuploidy (13).     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Linkage of Oxidative Stress and Mitochondrial Dysfunctions to Spontaneous Culture Degeneration in Aspergillus nidulans

Filamentous fungi including mushrooms frequently and spontaneously degenerate during subsequent culture maintenance on artificial media, which shows the loss or reduction abilities of asexual sporulation, sexuality, fruiting, and production of secondary metabolites, thus leading to economic losses during mass production. To better understand the underlying mechanisms of fungal degeneration, the model fungus Aspergillus nidulans was employed in this study for comprehensive analyses. First, linkage of oxidative stress to culture degeneration was evident in A. nidulans. Taken together with the verifications of cell biology and biochemical data, a comparative mitochondrial proteome analysis revealed that, unlike the healthy wild type, a spontaneous fluffy sector culture of A. nidulans demonstrated the characteristics of mitochondrial dysfunctions. Relative to the wild type, the features of cytochrome c release, calcium overload and up-regulation of apoptosis inducing factors evident in sector mitochondria suggested a linkage of fungal degeneration to cell apoptosis. However, the sector culture could still be maintained for generations without the signs of growth arrest. Up-regulation of the heat shock protein chaperones, anti-apoptotic factors and DNA repair proteins in the sector could account for the compromise in cell death. The results of this study not only shed new lights on the mechanisms of spontaneous degeneration of fungal cultures but will also provide alternative biomarkers to monitor fungal culture degeneration.Culture degeneration, also called colony deterioration, of filamentous fungi can frequently occur during subsequent maintenance of fungal culture on artificial media by showing morphological changes, such as colony sectorization, loss or impaired ability of sporulation, fruiting, and sexuality (1, 2). It was first called as “woolly degeneration” in the model fungus Neurospora crassa (3). These morphological variations are also accompanied with the loss or reduction in secondary metabolites production, thus resulting in great commercial losses (4–7). Different from the mutation of genes involved in conidiation producing fluffy phenotypes (8–10), fungal culture degeneration spontaneously occurs and is usually irreversible. The rate of colony deterioration varies considerably among fungal species/strains and correlates with the growth environments, especially the composition of nutrient medium (11, 12). In addition, unlike the phenotype of senescence observed in the model fungus Podospora anserina (13), morphological variants of other filamentous fungi could be subcultured for many generations without growth arrest (14, 15). The mechanism(s) underlying fungal culture degeneration is poorly understood. Genetic mutations were not evident in the degenerated fungal isolates of Penicillium chrysogenum (6). Otherwise, methylation of genomic DNA was reported in a sector of the plant pathogenic fungus Fusarium oxysporum after successive subculturing (16). In some fungi, degeneration was linked to chromosome instability (4, 17).Our previous studies on the insect pathogenic fungus Metarhizium anisopliae revealed that fungal culture degeneration showed the signs of aging such as cellular accumulation of reactive oxygen species (ROS)¹, mitochondrial (mt) dysfunctions, and mtDNA glycation (14, 15). According to the vicious cycle theory of aging, mitochondria are the primary source of intracellular ROS production and also one of the important targets of ROS damage, which leads to generation of additional ROS (18, 19). Mitochondrial proteomics analyses have been frequently performed for studies on human diseases, and the physiologies of plants and yeasts (20, 21). Proteins localized in the mitochondria control mt dynamics, morphology, and function and their dysregulation or damage may induce abnormality in mitochondrial function (22). However, it is not known whether these changes occur in degenerated fungal cultures.In this study, cell biology, biochemical and comparative mitochondrial proteomic analyses were performed by using the model fungus Aspergillus nidulans to better understand the features and mechanisms of fungal culture degeneration. We found a significant difference in mitochondrial protein profiles between the wild type (WT) and nonsporulation sector culture. Many of the altered proteins fall into the functional categories of energy metabolism, stress responses and cell death. Functional consequences of these changes are supported by our experimental data. The observed features such as cellular oxidative stress, mitochondrial dysfunctions, accelerated autophagy and releases of apoptotic factors in degenerated A. nidulans resemble the apoptotic process observed in mammalian cells.