Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

The exomer cargo adaptor structure reveals a novel GTPase‐binding domain

Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans‐Golgi network (TGN)‐derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (F N3–B RCT of e xomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes.     

Genetic Analysis of Yeast Sec24p Mutants Suggests Cargo Binding Is Not Co‐operative during ER Export

Many eukaryotic secretory proteins are selected for export from the endoplasmic reticulum (ER) through their interaction with the Sec24p subunit of the coat protein II (COPII) coat. Three distinct cargo‐binding sites on yeast Sec24p have been described by biochemical, genetic and structural studies. Each site recognizes a limited set of peptide motifs or a folded structural domain, however, the breadth of cargo recognized by a given site and the dynamics of cargo engagement remain poorly understood. We aimed to gain further insight into the broader molecular function of one of these cargo‐binding sites using a non‐biased genetic approach. We exploited the in vivo lethality associated with mutation of the Sec24p B‐site to identify genes that suppress this phenotype when overexpressed. We identified SMY2 as a general suppressor that rescued multiple defects in Sec24p, and SEC22 as a specific suppressor of two adjacent cargo‐binding sites, raising the possibility of allosteric regulation of these domains. We generated a novel set of mutations in Sec24p that distinguish these two sites and examined the ability of Sec22p to rescue these mutations. Our findings suggest that co‐operativity does not influence cargo capture at these sites, and that Sec22p rescue occurs via its function as a retrograde SNARE.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

粗糙脉孢菌木质纤维素降解利用研究进展简

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

Differential responses of wood-rot fungi cellulases towards polyclonal antibodies against Trichoderma viride cellobiohydrolase I

Cellobiohydrolase (CBH) I, a main component of Trichoderma extracellular protein, was purified to an electrophoretically homogeneous state from a commercial cellulase preparation (Meicelase from T. viride) by column chromatography on anion and cation exchangers. The difference in the cross-reactivity of cellulolytic enzyme systems of brown-rot and white-rot fungi with the polyclonal antibodies to the CBH I was studied by enzyme-linked immunosorbent assay (ELISA). The antibodies were observed to react quantitatively and with great sensitivity with the antigen (CBH I), and at the same time to cross-react to some extent with T. viride cellulase components other than the CBH I. Nevertheless, the intensity of cross-reactivity of wood-rot fungi cellulases with the antibodies was parallel to the activity of exo-1,4-ß-glucanase. The cellulase system from brown-rot fungi, believed to lack exo-1,4-ß-glucanases, gave a negative response towards the antibodies. These results suggested the presence of some homologous sequences and structures with the T. viride CBH I in the enzymes of white-rot fungi and their absence in those of brown-rot fungi. Correspondence to: M. Ishihara     

A comparative systems analysis of polysaccharide‐elicited responses in Neurospora crassa reveals carbon source‐specific cellular adaptations

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source‐specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat‐1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.     

The Rab GTPase YPT‐1 associates with Golgi cisternae and Spitzenkörper microvesicles in Neurospora crassa

Vesicle traffic involves budding, transport, tethering and fusion of vesicles with acceptor membranes. GTP‐bound small Rab GTPases interact with the membrane of vesicles, promoting their association with other factors before their subsequent fusion. Filamentous fungi contain at their hyphal apex the Spitzenkörper (Spk), a multivesicular structure to which vesicles concentrate before being redirected to specific cell sites. The regulatory mechanisms ensuring the directionality of the vesicles that travel to the Spk are still unknown. Hence, we analyzed YPT‐1, the Neurospora crassa homologue of Saccharomyces cerevisiae Ypt1p (Rab1), which regulates different secretory pathway events. Laser scanning confocal microscopy revealed fluorescently tagged YPT‐1 at the Spk and putative Golgi cisternae. Co‐expression of YPT‐1 and predicted post‐Golgi Rab GTPases showed YPT‐1 confined to the Spk microvesicular core, while SEC‐4 (Rab8) and YPT‐31 (Rab11) occupied the Spk macrovesicular peripheral layer, suggesting that trafficking and organization of macro and microvesicles at the Spk are regulated by distinct Rabs. Partial colocalization of YPT‐1 with USO‐1 (p115) and SEC‐7 indicated the additional participation of YPT‐1 at early and late Golgi trafficking steps.     

On‐site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip‐growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long‐range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three‐dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub‐apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short‐range “on‐site” secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.     

Trafficking of the myrosinase‐associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB

Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL‐like lipase implicated in glucosinolate metabolism through its association with the β‐glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild‐type plants suggested that GLL23 is normally post‐translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase‐associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi‐localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER–Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.     

MAIGO2 is involved in abscisic acid‐mediated response to abiotic stresses and Golgi‐to‐ER retrograde transport

The central role of multisubunit tethering complexes in intracellular trafficking has been established in yeast and mammalian systems. However, little is known about their roles in the stress responses and the early secretory pathway in Arabidopsis. In this study, Maigo2 (MAG2), which is equivalent to the yeast Tip20p and mammalian Rad50‐interacting protein, is found to be required for the responses to salt stress, osmotic stress and abscisic acid in seed germination and vegetative growth, and MAG2‐like (MAG2L) is partially redundant with MAG2 in response to environmental stresses. MAG2 strongly interacts with the central region of ZW10, and both proteins are important as plant endoplasmic reticulum (ER)‐stress regulators. ER morphology and vacuolar protein trafficking are unaffected in the mag2, mag2l and zw10 mutants, and the secretory marker to the apoplast is correctly transported in mag2 plants, which indicate that MAG2 functions as a complex with ZW10, and is potentially involved in Golgi‐to‐ER retrograde trafficking. Therefore, a new role for ER–Golgi membrane trafficking in abiotic‐stress and ER‐stress responses is discovered.     

A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI‐MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl‐CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.     

Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.     

Repeat-induced point (RIP) mutation in the industrial workhorse fungus Trichoderma reesei

Trichoderma reesei (syn. Hypocrea jecorina) is a filamentous ascomycete. Due to its capability of producing large amounts of lignocellulolytic enzymes and various heterologous proteins, this fungus has been widely used for industrial applications for over 70 years. It is also a model organism for lignocellulosic biomass degradation and metabolic engineering. Recently, we experimentally and computationally demonstrated that Trichoderma reesei exhibits high homology pairing and repeat-induced point (RIP) mutation activities at a premeiotic stage, i.e., between fertilization and karyogamy or premeiotic DNA replication. The discovery of RIP in Trichoderma reesei not only reveals significant impacts of sexual reproduction on evolution and chromosome architecture but also provides intriguing perspectives for industrial strain improvement. This review emphasizes two major points about RIP and RIP-like processes in Pezizomycotina fungi. First, the molecular mechanisms of RIP and RIP-like processes in Trichoderma reesei and other Pezizomycotina fungi are apparently distinct from those originally described in the model fungus Neurospora crassa. Second, orthologs of the rid1 (deficient in RIP-1) DNA methyltransferase gene were shown to be essential for sexual development in at least four Pezizomycotina fungi, including Trichoderma reesei. In contrast, rid1 is dispensable for Neurospora crassa sexual development. We suggest that the rid1-like gene products and/or their DNA methyltransferase activities play critical roles in promoting fungal sexual development. The Neurospora crassa rid1 gene might have lost this evolutionarily conserved function.

     

Investigating Endocytic Pathways to the Endoplasmic Reticulum and to the Cytosol Using SNAP‐Trap

Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP‐tag. Using ER‐targeted SNAP‐tag as reporter, we found that transport of CTB to the ER depends on dynamin‐2 and syntaxin 5. Plasma membrane proteins and a fluid‐phase marker added to the medium were also transported to the ER. This flux was not affected by exposing cells to CTB but was inhibited by depleting syntaxin 5 and increased by depleting dynamin‐2. As a control for confined intracellular localization of ER‐targeted SNAP‐tag we used adenovirus‐5, which traffics to endosomes and then escapes into the cytosol. The virus did not react with ER‐targeted SNAP but with cytosolic SNAP. Together, our results establish a new method (SNAP‐trap) to study trafficking of different cargo to the ER and the cytosol and provide evidence for the existence of a constitutive pathway from the cell surface to the ER .     

Synergism between components of the cellulase system of the anaerobic rumen fungus Neocallimastix frontalis and those of the aerobic fungi Penicillium pinophilum and Trichoderma koningii in degrading crystalline cellulose

The cellulase system of Neocallimastix frontalis was separated by differential affinity on cellulose into an adsorbed fraction that could solubilize crystalline cellulose (crystalline-cellulose-solubilizing fraction, CCSF), and a non-adsorbed fraction that contained endoglucanase and -glucosidase activities (non-adsorbed endoglucanase/ -glucosidas, NAE/-G) but which showed no activity to crystalline cellulose. Both fractions were tested for their capacity to act synergistically with the cellobiohydrolase (CBH) components of aerobic fungi in degrading crystalline cellulose. The CCSF acted synergistically with CBH I components of both Penicillium pinophilum and Trichoderma koningii but not with CBH II. The NAE/-G fraction also acted synergistically with the CBH components of P. pinophilum but, remarkably, only when both CBH I and CBH II were present in the reaction mixture. By comparison with previously published studies on the mechanism of action of P. pinophilum cellulase it is speculated that the CCSF of N. frontalis may contain CBH I- and CBH II-type enzymes.