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Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizocto... more Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea therefore providing an environmentally sound alternative to chemical fungicides in the management of these pathogens. Two-dimensional electrophoresis was used to display cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls. Protein profiles were compared to identify T. atroviride proteins up-regulated in the presence of theR. solani cell walls. Twenty four protein spots were identified using matrix assisted laser desorption ionisation mass spectrometry, liquid chromatography mass spectrometry and Nterminal sequencing. Three novel proteases to T. atroviride were up-regulated and identified as vacuolar serine protease, vacuolar protease A and trypsin-like protease. Two of these proteases, vacuolar protease A and vacuolar serine protease have been sequenced and cloned using chromosome walking PCR. Vacuolar protease A has two predicted introns, a predicted signal peptide and contains an aspartic proteinase conserved domain. Vacuolar serine protease has one predicted intron, a predicted signal peptide and contains a peptidase S8 conserved domain.1 page(s

SummaryWe have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyce... more SummaryWe have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyces cerevisiae chromosome XIV as part of the international Sc2.0 project. We showed that certain synthetic loxPsym recombination sites can interfere with mitochondrial protein localization, that the deletion of one intron (NOG2) reduced fitness, and that a reassigned stop codon can lead to a growth defect. In parallel to these rational debugging modifications, we used Adaptive Laboratory Evolution to generate a general growth defect suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss. The presence of wild-type chromosomes in the hybrid tetraploids increased post-SCRaMbLE viability and heterologous DNA integration, highlighting the plasticity of...

Bioresource Technology, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Background For the economic production of biofuels and other valuable products from lignocellulos... more Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes; with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene ( CEL3A ) from Saccharomycopsis fibuligera and an endoglucanase gene ( CEL5A ) from Trichoderma reesei . S. cerevisiae , transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that BPNPG5 (Megazyme®) is a suitable substrate to determine...

Algal Research, 2020

Contamination of the environment by heavy metals is an increasing problem globally. While the mec... more Contamination of the environment by heavy metals is an increasing problem globally. While the mechanisms bacteria and yeasts have developed to tolerate these metals are well covered in the published literature, much less is known about algae. In this study, the wild-type Euglena gracilis (Z-strain) and the sugar loving E. gracilis var. saccharophila (B-strain) were exposed to the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg). Minimum inhibitory concentration (MIC) studies indicated that the heavy metal tolerance of both strains was in the order of Pb > Cd > Hg. Based on microwave plasma atomic emission spectroscopy (MP-AES), the maximum Cd accumulation by Z-strain was 8.1 mg Cd per gram of dry weight (DW), which makes it a Cd hyperaccumulator. A total of 4493 proteins were quantified by SWATH mass spectrometry to assess the effect of the heavy metals on E. gracilis at the proteome level. In response to Cd, 960 proteins in the Z-strain and 127 in the B-strain changed in relative abundance compared to the untreated control. Proteins of high abundance included the major facilitator superfamily (MFS) transporters, cadmium/zinc-transporting ATPase and heavy metal transporting P1B-ATPase. Also, there was a substantial increase in the abundance of thiol-rich proteins that are paramount in metal chelation and sequestration as well as proteins involved in cellular stress response. A potential mechanism schematic for heavy accumulation in the E. gracilis Z-strain is outlined based on the data collected. The proteomic data presented here contribute to a better understanding of the effects of the exposure of E. gracilis to heavy metals by identifying proteins and thereby genes involved in heavy metal tolerance and accumulation. The information obtained can eventually be utilized for generating highly heavy metal-tolerant Euglena strains for environmental applications. The most common heavy metal pollutants are lead (Pb), cadmium (Cd), mercury (Hg), nickel (Ni), chromium (Cr), arsenic (As), zinc (Zn) and copper (Cu). Heavy metals can attach to the metal-binding sites of proteins such as sulfhydryl groups and disrupt cellular functions resulting in the increase of reactive oxygen species (ROS), thus causing cellular stress and apoptosis [5,6]. The current study focuses on Cd, Pb and Hg, the three most toxic heavy metals of which very low amounts can to cause serious health problems for humans [7-9]. Exposure to Cd and Pb has been linked to kidney and bone damage, reproductive failure, cancer, and damage to the central nervous and immune systems [7,10,11]. Likewise, Hg exposure results in damage to the nervous system, heart disease and kidney damage [12].

Scientific Reports, 2019

Scedosporium fungi are found in various natural and host-associated environments, including the l... more Scedosporium fungi are found in various natural and host-associated environments, including the lungs of cystic fibrosis patients. However, their role in infection development remains underexplored. Here the attachment of conidia of a virulent S. aurantiacum strain WM 06.482 onto the human lung epithelial A549 cells in vitro was visualized using microscopy to examine the initial steps of infection. We showed that 75–80% of fungal conidia were bound to the A549 cells within four hours of co-incubation, and started to produce germ tubes. The germinating conidia seemed to invade the cells through the intercellular space, no intracellular uptake of fungal conidia by the airway epithelial cells after conidial attachment. Transcriptomic analysis of the A549 cells revealed that the up-regulated genes were mainly associated with cell repair and inflammatory processes indicating a protective response against S. aurantiacum infection. Network analysis of the differentially expressed genes sho...

Algal Research, 2019

Euglena gracilis can use a wide range of organic carbon sources, as well as CO 2 from the atmosph... more Euglena gracilis can use a wide range of organic carbon sources, as well as CO 2 from the atmosphere. This metabolic versatility is owed to the genome of E. gracilis that can encode a wide range of enzymes. Many of these enzymes are regulated post-transcriptionally, allowing the cells to adapt quickly to changes in their surroundings. Here we investigated the effect of predominantly phototrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on central carbon metabolism in E. gracilis Z using label-free shotgun proteomics. Differential expression between isozymes was observed based on the cultivation condition. A hexokinase enzyme identified in the published transcriptome was not detected in the proteome. Instead, a high-specificity glucokinase appeared to conduct the first step of glycolysis. Two candidates for paramylon synthase were identified (EgGSL1 and EgGSL2), of which the predominant EgGSL2 protein was detected across all growth conditions, while EgGSL1 was only detected in the presence of light (PT and MT cultivations). Proteomic analysis revealed that the oxidative pentose phosphate pathway also plays a key role in glucose metabolism under MT and HT cultivation. Some chloroplast-encoded proteins and enzymes of the Calvin pathway were detected under HT cultivation indicating regulation at the post-translational level. The carbon metabolic pathways investigated here in terms of proteomic changes provide new information, as well as validate data presented elsewhere with quantitative proteomics, adding to the existing knowledge of metabolism in E. gracilis. Putative functional annotations of several proteins that were previously unidentified are also provided.

Algal Research, 2019

Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmece... more Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.

Metarhizium anisopliae is a naturally occurring biological control of many insects including the ... more Metarhizium anisopliae is a naturally occurring biological control of many insects including the greyback canegrub (Dermolepida albohirtum), a sugarcane pest in Australia. While there have been some gene-based approaches into identifying determinants for biological control and developing improved strains, our study provides a comparative proteomics approach into identifying key proteins produced by Metarhizium anisopliae during infection of greyback canegrubs. At the same time, we have developed a proteomic map for the greyback canegrub larvae responsible for significant losses of the crop. Pathogenicity related proteins were identified by both a liquid culture and solid culture approach. Solid culture approach is thought to give a more realistic view of infection process compared to liquid culture. In order to identify novel target proteins, differential displays of proteomic maps of healthy/infected cuticle (HC/IC) as well as healthy/infected whole grub (HWG/IWG) were produced and analysed using Progenesis image analysis software. Proteomic map of healthy fungus (Metarhizium anisopliae) (MY) was also generated successfully. A total of 156 protein spots on HWG, 88 unique spots on IWG, 17 spots from HC and 14 spots from MY were analysed by mass spectrometry. Of these, 61 protein spots from HWG, 40 protein spots from IWG, 15 protein spots from HC and 9 protein spots from MY were confirmed by identification using mass spectrometry. Among the identified proteins were different forms of actin and tropomyosin, an ATP binding protein, arginine kinase, formate dehydrogenalase, enolase, tara like protein isoform and heat shock proteins. Further identification for most of the protein spots has been hindered due to the limited number of suitable/accessible databases. Metarhizium anisopliae has been successfully transformed to benomyl resistance using pBENA3, a plasmid containing the benA3 allele from Aspergillus nidulans using particle bombardment as a preparation for introducing genes encoding the identified pathogenesis factors into Metarhizium.1 page(s

The filamentous fungus Trichoderma reesei is one of the most efficient eukaryotic cell factories ... more The filamentous fungus Trichoderma reesei is one of the most efficient eukaryotic cell factories available. Considering its extraordinary secretion capacity, this species can be characterised as “a professional” protein secretor. Protein quality control is a crucial cellular function. Proteins that are not folded correctly or not fully assembled are recognised in the early secretory pathway and subjected to degradation by the ubiquitin-proteasome pathway featuring a large (approximately 2.5 MDa) multicatalytic protease, the proteasome. The proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated process. An isolation method for the 20S proteasome of T. reesei and a 2D master map of the fungal proteasome have been established. From the map, a number of proteasome subunit proteins as well as proteasome-interacting proteins have been identified. We are also in the process of creating a series of mutant forms of the main cellobiohydrolase enzyme CBHI to trace its secretion and presumed degradation in the proteasome.1 page(s

Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizocto... more Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea therefore providing an environmentally sound alternative to chemical fungicides in the management of these pathogens. Two-dimensional electrophoresis was used to display cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls. Protein profiles were compared to identify T. atroviride proteins up-regulated in the presence of theR. solani cell walls. Twenty four protein spots were identified using matrix assisted laser desorption ionisation mass spectrometry, liquid chromatography mass spectrometry and Nterminal sequencing. Three novel proteases to T. atroviride were up-regulated and identified as vacuolar serine protease, vacuolar protease A and trypsin-like protease. Two of these proteases, vacuolar protease A and vacuolar serine protease have been sequenced and cloned using chromosome walking PCR. Vacuolar protease A has two predicted introns, a predicted signal peptide and contains an aspartic proteinase conserved domain. Vacuolar serine protease has one predicted intron, a predicted signal peptide and contains a peptidase S8 conserved domain.1 page(s

SummaryWe have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyce... more SummaryWe have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyces cerevisiae chromosome XIV as part of the international Sc2.0 project. We showed that certain synthetic loxPsym recombination sites can interfere with mitochondrial protein localization, that the deletion of one intron (NOG2) reduced fitness, and that a reassigned stop codon can lead to a growth defect. In parallel to these rational debugging modifications, we used Adaptive Laboratory Evolution to generate a general growth defect suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss. The presence of wild-type chromosomes in the hybrid tetraploids increased post-SCRaMbLE viability and heterologous DNA integration, highlighting the plasticity of...

Bioresource Technology, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Background For the economic production of biofuels and other valuable products from lignocellulos... more Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes; with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene ( CEL3A ) from Saccharomycopsis fibuligera and an endoglucanase gene ( CEL5A ) from Trichoderma reesei . S. cerevisiae , transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that BPNPG5 (Megazyme®) is a suitable substrate to determine...

Algal Research, 2020

Contamination of the environment by heavy metals is an increasing problem globally. While the mec... more Contamination of the environment by heavy metals is an increasing problem globally. While the mechanisms bacteria and yeasts have developed to tolerate these metals are well covered in the published literature, much less is known about algae. In this study, the wild-type Euglena gracilis (Z-strain) and the sugar loving E. gracilis var. saccharophila (B-strain) were exposed to the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg). Minimum inhibitory concentration (MIC) studies indicated that the heavy metal tolerance of both strains was in the order of Pb > Cd > Hg. Based on microwave plasma atomic emission spectroscopy (MP-AES), the maximum Cd accumulation by Z-strain was 8.1 mg Cd per gram of dry weight (DW), which makes it a Cd hyperaccumulator. A total of 4493 proteins were quantified by SWATH mass spectrometry to assess the effect of the heavy metals on E. gracilis at the proteome level. In response to Cd, 960 proteins in the Z-strain and 127 in the B-strain changed in relative abundance compared to the untreated control. Proteins of high abundance included the major facilitator superfamily (MFS) transporters, cadmium/zinc-transporting ATPase and heavy metal transporting P1B-ATPase. Also, there was a substantial increase in the abundance of thiol-rich proteins that are paramount in metal chelation and sequestration as well as proteins involved in cellular stress response. A potential mechanism schematic for heavy accumulation in the E. gracilis Z-strain is outlined based on the data collected. The proteomic data presented here contribute to a better understanding of the effects of the exposure of E. gracilis to heavy metals by identifying proteins and thereby genes involved in heavy metal tolerance and accumulation. The information obtained can eventually be utilized for generating highly heavy metal-tolerant Euglena strains for environmental applications. The most common heavy metal pollutants are lead (Pb), cadmium (Cd), mercury (Hg), nickel (Ni), chromium (Cr), arsenic (As), zinc (Zn) and copper (Cu). Heavy metals can attach to the metal-binding sites of proteins such as sulfhydryl groups and disrupt cellular functions resulting in the increase of reactive oxygen species (ROS), thus causing cellular stress and apoptosis [5,6]. The current study focuses on Cd, Pb and Hg, the three most toxic heavy metals of which very low amounts can to cause serious health problems for humans [7-9]. Exposure to Cd and Pb has been linked to kidney and bone damage, reproductive failure, cancer, and damage to the central nervous and immune systems [7,10,11]. Likewise, Hg exposure results in damage to the nervous system, heart disease and kidney damage [12].

Scientific Reports, 2019

Scedosporium fungi are found in various natural and host-associated environments, including the l... more Scedosporium fungi are found in various natural and host-associated environments, including the lungs of cystic fibrosis patients. However, their role in infection development remains underexplored. Here the attachment of conidia of a virulent S. aurantiacum strain WM 06.482 onto the human lung epithelial A549 cells in vitro was visualized using microscopy to examine the initial steps of infection. We showed that 75–80% of fungal conidia were bound to the A549 cells within four hours of co-incubation, and started to produce germ tubes. The germinating conidia seemed to invade the cells through the intercellular space, no intracellular uptake of fungal conidia by the airway epithelial cells after conidial attachment. Transcriptomic analysis of the A549 cells revealed that the up-regulated genes were mainly associated with cell repair and inflammatory processes indicating a protective response against S. aurantiacum infection. Network analysis of the differentially expressed genes sho...

Algal Research, 2019

Euglena gracilis can use a wide range of organic carbon sources, as well as CO 2 from the atmosph... more Euglena gracilis can use a wide range of organic carbon sources, as well as CO 2 from the atmosphere. This metabolic versatility is owed to the genome of E. gracilis that can encode a wide range of enzymes. Many of these enzymes are regulated post-transcriptionally, allowing the cells to adapt quickly to changes in their surroundings. Here we investigated the effect of predominantly phototrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on central carbon metabolism in E. gracilis Z using label-free shotgun proteomics. Differential expression between isozymes was observed based on the cultivation condition. A hexokinase enzyme identified in the published transcriptome was not detected in the proteome. Instead, a high-specificity glucokinase appeared to conduct the first step of glycolysis. Two candidates for paramylon synthase were identified (EgGSL1 and EgGSL2), of which the predominant EgGSL2 protein was detected across all growth conditions, while EgGSL1 was only detected in the presence of light (PT and MT cultivations). Proteomic analysis revealed that the oxidative pentose phosphate pathway also plays a key role in glucose metabolism under MT and HT cultivation. Some chloroplast-encoded proteins and enzymes of the Calvin pathway were detected under HT cultivation indicating regulation at the post-translational level. The carbon metabolic pathways investigated here in terms of proteomic changes provide new information, as well as validate data presented elsewhere with quantitative proteomics, adding to the existing knowledge of metabolism in E. gracilis. Putative functional annotations of several proteins that were previously unidentified are also provided.

Algal Research, 2019

Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmece... more Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.

Metarhizium anisopliae is a naturally occurring biological control of many insects including the ... more Metarhizium anisopliae is a naturally occurring biological control of many insects including the greyback canegrub (Dermolepida albohirtum), a sugarcane pest in Australia. While there have been some gene-based approaches into identifying determinants for biological control and developing improved strains, our study provides a comparative proteomics approach into identifying key proteins produced by Metarhizium anisopliae during infection of greyback canegrubs. At the same time, we have developed a proteomic map for the greyback canegrub larvae responsible for significant losses of the crop. Pathogenicity related proteins were identified by both a liquid culture and solid culture approach. Solid culture approach is thought to give a more realistic view of infection process compared to liquid culture. In order to identify novel target proteins, differential displays of proteomic maps of healthy/infected cuticle (HC/IC) as well as healthy/infected whole grub (HWG/IWG) were produced and analysed using Progenesis image analysis software. Proteomic map of healthy fungus (Metarhizium anisopliae) (MY) was also generated successfully. A total of 156 protein spots on HWG, 88 unique spots on IWG, 17 spots from HC and 14 spots from MY were analysed by mass spectrometry. Of these, 61 protein spots from HWG, 40 protein spots from IWG, 15 protein spots from HC and 9 protein spots from MY were confirmed by identification using mass spectrometry. Among the identified proteins were different forms of actin and tropomyosin, an ATP binding protein, arginine kinase, formate dehydrogenalase, enolase, tara like protein isoform and heat shock proteins. Further identification for most of the protein spots has been hindered due to the limited number of suitable/accessible databases. Metarhizium anisopliae has been successfully transformed to benomyl resistance using pBENA3, a plasmid containing the benA3 allele from Aspergillus nidulans using particle bombardment as a preparation for introducing genes encoding the identified pathogenesis factors into Metarhizium.1 page(s

The filamentous fungus Trichoderma reesei is one of the most efficient eukaryotic cell factories ... more The filamentous fungus Trichoderma reesei is one of the most efficient eukaryotic cell factories available. Considering its extraordinary secretion capacity, this species can be characterised as “a professional” protein secretor. Protein quality control is a crucial cellular function. Proteins that are not folded correctly or not fully assembled are recognised in the early secretory pathway and subjected to degradation by the ubiquitin-proteasome pathway featuring a large (approximately 2.5 MDa) multicatalytic protease, the proteasome. The proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated process. An isolation method for the 20S proteasome of T. reesei and a 2D master map of the fungal proteasome have been established. From the map, a number of proteasome subunit proteins as well as proteasome-interacting proteins have been identified. We are also in the process of creating a series of mutant forms of the main cellobiohydrolase enzyme CBHI to trace its secretion and presumed degradation in the proteasome.1 page(s