Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe

The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.     

The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase (P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.     

Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery

Protein misfolding is monitored by a variety of cellular "quality control" systems. Endoplasmic reticulum (ER) quality control handles misfolded secretory and membrane proteins and is well characterized. However, less is known about the quality control of misfolded cytosolic proteins (CytoQC). To study CytoQC, we have employed a genetic system in Saccharomyces cerevisiae using a transplantable degron, CL1 (1). Attachment of CL1 to the cytosolic protein Ura3p destabilizes Ura3p, targeting it for rapid proteasomal degradation. We have performed a comprehensive analysis of Ura3p-CL1 degradation requirements. As shown previously, we observe that the ER-localized ubiquitin E2 (Ubc6p, Ubc7p, and Cue1p) and E3 (Doa10p) machinery involved in ER-associated degradation (ERAD) are also responsible for the degradation of the cytosolic substrate Ura3p-CL1. Importantly, we find that the cytosol/ER membrane-localized chaperones Ydj1p and Ssa1p, known to be necessary for the ERAD of membrane proteins with misfolded cytosolic domains, are also required for the ubiquitination and degradation of Ura3p-CL1. In addition, we show a role for the Cdc48p-Npl4p-Ufd1p complex in the degradation of Ura3p-CL1. When ubiquitination is blocked, a portion of Ura3p-CL1 is ER membrane-localized. Furthermore, access to the cytosolic face of the ER is required for the degradation of CL1 degron-containing proteins. The ER is distributed throughout the cytosol, and our data, together with previous studies, suggest that the cytosolic face of the ER membrane serves as a "platform" for the degradation of Ura3p-CL1, which may also be the case for other CytoQC substrates.     

Filament formation by metabolic enzymes—A new twist on regulation

Compartmentalization of metabolic enzymes through protein–protein interactions is an emerging mechanism for localizing and regulating metabolic activity. Self-assembly into linear filaments is a common strategy for cellular compartmentalization of enzymes. Polymerization is often driven by changes in the metabolic state of the cell, suggesting that it is a strategy for shifting metabolic flux in response to cellular demand. Although polymerization of metabolic enzymes is widespread, observed from bacteria to humans, we are just beginning to appreciate their role in regulating cellular metabolism. In most cases, one functional role of metabolic enzyme filaments is allosteric control of enzyme activity. Here, we highlight recent findings, providing insight into the structural and functional significance of filamentation of metabolic enzymes in cells.     

Role of nucleotide binding in septin-septin interactions and septin localization in Saccharomyces cerevisiae

Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins.     

Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae

We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.     

Different polypeptides form the intermediate filaments in bovine hoof and esophageal epithelium and in aortic endothelium

Polypeptides that form 10-nm filaments in vitro were isolated from three different bovine tissues: the viable portion of the hoof epithelium, the epithelium of the esophagus, and cultured endothelial cells derived from aorta. The seven polypeptides from hoof, the two from esophagus, and the one from endothelial cells were different with respect to mobility in SDS polyacrylamide gels and/or limited proteolytic digestion. Peptide maps of the different filament-forming polypeptides (FFP's) showed that none of the smaller FFP's was a fragment of any of the larger FFP's. Several isomobile fragments were found in the peptide maps of different FFP's, suggesting that they might contain regions of amino acid sequence homology. We present a hypothesis that suggests how the different 10-nm filament-forming proteins may be related.     

GTP binding is required for SEPT12 to form filaments and to interact with SEPT11

Septins are a family of filament-forming GTP-binding proteins involved in a variety of cellular process such as cytokinesis, exocytosis, and membrane dynamics. Here we report the biochemical and immunocytochemical characterization of a recently identified mammalian septin, SEPT12. SEPT12 binds GTP in vitro, and a mutation (Gly56 to Asn) in the GTP-binding motif abolished binding. Immunocytochemical analysis revealed that wild-type SEPT12 formed filamentous structures when transiently expressed in Hela cells whereas SEPT12G56A generated large aggregates. In addition, wild-type SEPT12 failed to form filaments when coexpressed with SEPT12G56A. We also observed that GTP-binding by SEPT12 is required for interaction with SEPT11 but not with itself.     

Degradation signals recognized by the Ubc6p-Ubc7p ubiquitin-conjugating enzyme pair

Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of beta-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Delta and sec61-2) did not inhibit the degradation of the beta-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p.     

Phylogenetic and Preliminary Phenotypic Analysis of Yeast PAQR Receptors: Potential Antifungal Targets

Proteins belonging to the Progestin and AdipoQ Receptor (PAQR) superfamily of membrane bound receptors are ubiquitously found in fungi. Nearly, all fungi possess two evolutionarily distinct paralogs of PAQR protein, which we have called the PQRA and PQRB subtypes. In the model fungus Saccharomyces cerevisiae, these subtypes are represented by the Izh2p and Izh3p proteins, respectively. S. cerevisiae also possesses two additional PQRA-type receptors called Izh1p and Izh4p that are restricted to other species within the "Saccharomyces complex". Izh2p has been the subject of several recent investigations and is of particular interest because it regulates fungal growth in response to proteins produced by plants and, as such, represents a new paradigm for interspecies communication. We demonstrate that IZH2 and IZH3 gene dosage affects resistance to polyene antifungal drugs. Moreover, we provide additional evidence that Izh2p and Izh3p negatively regulate fungal filamentation. These data suggest that agonists of these receptors might make antifungal therapeutics, either by inhibiting fungal development or by sensitizing fungi to the toxic effects of current antifungal therapies. This is particularly relevant for pathogenic fungi such as Candida glabrata that are closely related to S. cerevisiae and contain the same complement of PAQR receptors.     

Blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in Cryptococcus neoformans

Cryptococcus neoformans is a heterothallic basidiomycetous yeast that primarily infects immunocompromised individuals. Dikaryotic hyphae resulting from the fusion of the MATa and MATalpha mating type strains represent the filamentous stage in the sexual life cycle of C. neoformans. In this study we demonstrate that the production of dikaryotic filaments is inhibited by blue light. To study blue light photoresponse in C. neoformans, we have identified and characterized two genes, CWC1 and CWC2, which are homologous to Neurospora crassa wc-1 and wc-2 genes. Conserved domain analyses indicate that the functions of Cwc1 and Cwc2 proteins may be evolutionally conserved. To dissect their roles in the light response, the CWC1 gene deletion mutants are created in both mating type strains. Mating filamentation in the bilateral cross of cwc1 MATa and MATalpha strains is not sensitive to light. The results indicate that Cwc1 may be an essential regulator of light responses in C. neoformans. Furthermore, overexpression of the CWC1 or CWC2 gene requires light activation to inhibit sexual filamentation, suggesting both genes may function together in the early step of blue light signalling. Taken together, our findings illustrate blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans.     

Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.     

Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs

Candida albicans is an important commensal of mucosal surfaces that is also an opportunistic pathogen. This organism colonizes a wide range of host sites that differ in pH; thus, it must respond appropriately to this environmental stress to survive. The ability to respond to neutral-to-alkaline pHs is governed in part by the RIM101 signal transduction pathway. Here we describe the analysis of C. albicans Rim13p, a homolog of the Rim13p/PalB calpain-like protease member of the RIM101/pacC pathway from Saccharomyces cerevisiae and Aspergillus nidulans, respectively. RIM13, like other members of the RIM101 pathway, is required for alkaline pH-induced filamentation and growth under extreme alkaline conditions. Further, our studies suggest that the RIM101 pathway promotes pH-independent responses, including resistance to high concentrations of lithium and to the drug hygromycin B. RIM13 encodes a calpain-like protease, and we found that Rim101p undergoes a Rim13p-dependent C-terminal proteolytic processing event at neutral-to-alkaline pHs, similar to that reported for S. cerevisiae Rim101p and A. nidulans PacC. However, we present evidence that suggests that C. albicans Rim101p undergoes a novel processing event at acidic pHs that has not been reported in either S. cerevisiae or A. nidulans. Thus, our results provide a framework to understand how the C. albicans Rim101p processing pathway promotes alkaline pH-independent processes.     

Cell Envelope Components Influencing Filament Length in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.