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Applied and Environmental Microbiology, August 2005, p. 4856-4861, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4856-4861.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

Mamoru Ohneda, Manabu Arioka, and Katsuhiko Kitamoto^*

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 27 August 2004/ Accepted 11 March 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.

Top Abstract Introduction Materials and Methods Results Discussion References

 
For the budding yeast Saccharomyces cerevisiae, more than 50 vacuolar protein sorting mutants, which missort and secrete vacuolar proteins such as carboxypeptidase Y (CPY) into the medium, have been isolated (3, 23, 24). Based on morphological characterizations, these mutants have been placed into six classes, classes A, B, C, D, E, and F (21). Some of the vacuolar protein sorting (VPS) proteins that belong to class B or C are involved in vacuolar biogenesis (4). In class E mutants, CPY accumulates in a deformed prevacuolar compartment, suggesting that class E VPS proteins are involved in transport of CPY from prevacuolar compartments to vacuoles (21). VPS proteins also are involved in other vacuolar transport pathways, such as those for alkaline phosphatase, cytoplasm-to-vacuole targeting, and endocytic pathways (2, 5, 18).

In animals and plants, homologs of several VPS genes have been isolated and characterized (10, 25, 28). However, systematic studies of vacuolar protein sorting and vacuolar morphogenesis have not been conducted with filamentous fungi, except for a few reports referring to mutants or genes related to vacuolar morphogenesis in Aspergillus nidulans. For instance, one cycloheximide-sensitive mutant of A. nidulans shows fragmentation of vacuoles (6). Geissenhoner et al. (8) showed that mutation of digA, a homolog of the S. cerevisiae VPS18/PEP3 locus, causes fragmented vacuoles and clustered mitochondria and nuclei, suggesting that the protein functions in organellar positioning and cell polarity in A. nidulans. Previously, we isolated the vpsA, avaA, and avaB genes (homologs of S. cerevisiae VPS1, VAM4/YPT7, and VAM6/VPS39, respectively) from A. nidulans and demonstrated that these genes are involved in vacuolar biogenesis (15, 17, 26). We also cloned cpyA, a homolog of S. cerevisiae PRC1 encoding a CPY precursor, from A. nidulans and showed that CpyA is the intracellular carboxypeptidase (16).

Aspergillus oryzae is an important microorganism in the Japanese fermentation industry. It can secrete large amounts of protein into the medium and has been used to produce both homologous and heterologous proteins (20). Elucidation of the molecular mechanisms of intracellular protein trafficking could improve the utilization of A. oryzae for the production of commercial enzymes. A. oryzae also has basic biological characteristics, such as filamentous growth, hyphal branching, asexual reproductive structures, and multicellularity, that cannot be studied in unicellular microorganisms such as S. cerevisiae.

Recently, we developed a system for monitoring the behavior of a vacuolar protein by expressing CpyA fused with enhanced green fluorescent protein (EGFP) (CPY-EGFP) in A. oryzae (14). CPY-EGFP was correctly transported to vacuoles, although the intensity of EGFP fluorescence in vacuoles may be affected by the vacuolar pH since EGFP fluoresces poorly and may be degraded by low-pH-activated vacuolar proteases (14). We predicted that it would be possible to isolate vps mutants of A. oryzae by using the CPY-EGFP expression system and looking for either an increase in extracellular EGFP fluorescence or a decrease in intracellular EGFP fluorescence caused by missorting of CPY-EGFP. These mutants could be identified by fluorescence-activated cell sorting (FACS) in order to identify conidia that do not fluoresce normally (11) due to missorting of CPY-EGFP. A fluorescence microplate reader can be used to identify A. oryzae vacuolar protein sorting mutants that missort and secrete CPY-EGFP. These high-throughput analyzers enable screening for A. oryzae vacuolar protein sorting mutants.

Our objectives in this study were to isolate vacuolar protein sorting mutants of A. oryzae and to use these mutants to identify previously undescribed membrane traffic mechanisms in filamentous fungi. We assumed that the levels of intracellular vacuolar proteins would decrease and the levels of extracellular vacuolar proteins would increase in vacuolar protein sorting mutants and used these properties to isolate such mutants. The phenotypes of the mutants suggest that vacuolar protein sorting, pH response, and hyphal morphogenesis are correlated in A. oryzae and provide new insights into the vacuolar protein sorting mechanism of filamentous fungi at a molecular level.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A. oryzae strains and growth conditions.
A. oryzae strains expressing CPY-EGFP were constructed by transformation of NS4 (niaD^– sC^–) with pCESC (sC cpyA-egfp) (14). An sC⁺ transformant designated NSCE1 was selected as the parent strain for mutagenesis and the control strain for analyses. For microscopic observation, extraction of total protein, and measurement of EGFP fluorescence in the medium, A. oryzae was cultivated in 100 ml of M medium (2 g/liter NH₄Cl, 1 g/liter NH₃SO₄, 0.5 g/liter KCl, 0.5 g/liter NaCl, 1 g/liter KH₂PO₄, 0.5 g/liter MgCl₂ · 7H₂O, 20 mg/liter FeSO₄, 20 g/liter glucose; pH 5.5 or 8.0) in a 500 ml-flask with shaking at 150 rpm at 30°C. For colony formation, conidiation, and measurement of the growth rate, A. oryzae was incubated statically at 30°C on M medium containing 1.5 g/liter agar.

Mutagenesis of A. oryzae conidia by UV irradiation.
Conidia of NSCE1 expressing CPY-EGFP were harvested from 5-day-old colonies on agar-containing M medium (pH 5.5) and were suspended in 0.01% Tween 80 at a concentration of 4 x 10⁵ conidia/ml. Five milliliters of the conidial suspension was poured into a sterilized petri dish (diameter, 90 mm). The conidia were irradiated in the dish for 5 min using brand new UV lamps (two 15-W lamps; wavelength, 260 nm) in a safety cabinet (SCV-ECIIA; Hitachi, Tokyo, Japan) at a distance of 60 cm, which resulted in approximately 1% survival. Under these conditions, the energy density irradiated to conidia was 40 µW/cm². Before analysis by FACS, approximately 20,000 conidia were spread on agar-containing M medium at a density of 200 survivors/plate. Conidia harvested from the colonies grown for 5 days at 30°C were screened for mutants. For fluorescence microplate reader (Spectrafluo plus or SAFIRE; Tecan, Mannedorg, Switzerland) screening, UV-irradiated conidia were inoculated directly into M medium in the wells of 96-well microplates.

Isolation of A. oryzae mutants showing dim EGFP fluorescence in conidia (dfc).
Conidia of NSCE1 expressing CPY-EGFP were mutagenized by UV irradiation and cultivated for conidiation. Conidia of the mutants were harvested and analyzed by FACS to isolate conidia with dim fluorescence. After repeated sorting and conidiation, single colonies were isolated and cultivated separately. Colonies that formed significantly higher numbers of conidia with less intense fluorescence than NSCE1 were designated dfc mutants.

Isolation of A. oryzae mutants showing hyper-EGFP fluorescence released into the medium (hfm).
Conidia of NSCE1 were mutagenized by UV irradiation and inoculated into 96-well microplates at an average density of 10 viable conidia/well. After cultivation, the EGFP fluorescence of the culture medium was measured with a fluorescent microplate reader (primary screen). The hyphal mixtures showing hyper-EGFP fluorescence released into the medium were plated and cultivated for conidiation. Conidia harvested from them were inoculated into 96-well microplates at a density of less than one conidium/well. After cultivation, EGFP fluorescence released into the medium was measured again (secondary screen).

Analysis of A. oryzae conidia by FACS.
Conidia harvested from 5-day-old colonies of A. oryzae were suspended in distilled water at a concentration of 10⁶ conidia/ml. A flow cytometer (FACSCalibur; Becton Dickinson Biosciences, San Jose, CA) operated using Cell Quest version 3.1 (Becton Dickinson Biosciences) was used for measurement of EGFP fluorescence in conidia and for sorting conidia with dim fluorescence. The FACS method used was that described by Maruyama et al. (11).

Measurement of EGFP fluorescence released into the medium with a spectrofluorometer.
Conidia of A. oryzae were cultured in 100 ml of M medium (pH 5.5 or 8.0) in a 500-ml flask by using an inoculation density of 10⁴ conidia/ml and were incubated at 30°C with shaking at 150 rpm for 3 days. The culture medium was sampled during the log phase of growth to reduce the amount of extracellular EGFP fluorescence due to cell lysis. Culture medium filtered through a 0.20-µm filter (Sartorius, Göttingen, Germany) was used as the spent medium for analyses. The spent medium was buffered to 0.1 M (final concentration) Tris-HCl (pH 8.0), and EGFP fluorescence was measured using a spectrofluorometer (FP-6500; Jasco, Tokyo, Japan) with 488 nm as the excitation wavelength and 510 nm as the emission wavelength at the high-sensitivity setting.

Observation of A. oryzae hyphae by fluorescence microscopy.
Conidia of A. oryzae were suspended in M medium at a density of 10⁴ conidia/ml and were incubated at 30°C with shaking for 24 h. Actively growing hyphae were observed by differential interference contrast (DIC) and fluorescence microscopy (BX52; Olympus, Tokyo, Japan) using MetaMorph 4.5f6 (Universal Imaging, Downingtown, PA). For staining with 7-amino-4-chloromethylcoumarin (CMAC) (Molecular Probes, Eugene, OR), hyphae were incubated in fresh M medium containing 10 µM CMAC at 30°C for 30 min and then washed in fresh M medium at 37°C for 30 min before observation by fluorescence microscopy. By observation of CMAC-stained A. oryzae expressing EGFP in the cytosol, it was confirmed that there is no cross talk between EGFP and CMAC fluorescence. At least 70 cells in different observation fields were evaluated for each observation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutants with reduced EGFP fluorescence in conidia.
There is a positive correlation between the intensity of EGFP fluorescence and conidium size (i.e., larger conidia are brighter), so the region from which conidia with dim fluorescence were sorted was adjusted accordingly. After mutagenesis, FACS, and conidial isolation, a slight increase in the number of dim conidia present was observed compared to nonmutagenized conidia of the parental strain (Table 1). The number of dim conidia increased significantly after repeated cycles of sorting and conidial isolation (Table 1). After three rounds of sorting and conidial isolation, 50 single colonies were selected for further analysis. The percentage of dfc conidia from 20 of these 50 colonies was more than twice as high as that from NSCE1 when the number of dfc conidia was counted by FACS analyses three times. The mutants were designated dfc mutants, and their phenotype was confirmed by fluorescence microscopy (Fig. 1). The distribution of fluorescence among dfc mutants varied. For example, the EGFP fluorescence was very dim throughout the cytoplasm of conidia of the dfc-1, dfc-6, and dfc-16 mutants, while the number of fluorescent spots in the dfc-4, dfc-11, and dfc-14 mutants was lower.

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TABLE 1. Increase in the number of dim conidia after UV irradiation mutagenesis and repeated sorting

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FIG. 1. Isolation of A. oryzae mutants showing dim EGFP fluorescence in conidia (dfc). Conidia harvested from NSCE1 and different mutants were observed by fluorescence microscopy. Bar, 5 µm.

Isolation of mutants with hyper-EGFP fluorescence.
Increased EGFP fluorescence compared to that of the NSCE1 parent was observed in the media of 20 of 1,920 wells screened. Single colonies were isolated from these wells, and mutants exhibiting intense EGFP fluorescence in the media were obtained. A total of 22 mutants were isolated and designated hfm (hyper-EGFP fluorescence released into the medium) mutants, including two mutants (hfm-21 and hfm-22) chosen from 2,000 mutated conidia evaluated with a different fluorescence microplate reader.

Missorting of vacuolar proteins into the media.
All of the mutants had growth rates similar to that of the wild type at both pH 5.5 and 8.0 when the wet weights of 1- to 3-day-old hyphae in submerged cultures and the diameters of 1- to 5-day-old colonies in the agar plate cultures were measured. Thirteen of the dfc mutants and all of the hfm mutants exhibited higher levels of EGFP fluorescence in the culture media than the parental strain exhibited. These 35 mutants could have included vps mutants.

In the four mutants with the highest levels of EGFP fluorescence compared to the parent strain, dfc-14, hfm-4, hfm-7, and hfm-21, the EGFP fluorescence was pH dependent (Table 2). dfc-14 and hfm-4 grown at pH 5.5 had approximately 12-fold-higher EGFP fluorescence in the culture media than the parent strain, but at pH 8.0 the difference was only twofold. In contrast, for hfm-21 the EGFP fluorescence released into the medium was approximately threefold higher than that of the parent strain at pH 5.5, but the fluorescence was 35-fold higher than that of the parent strain at pH 8.0. For hfm-7 the EGFP fluorescence released into the medium was fourfold and sixfold higher than that of the parent at pH 5.5 and 8.0, respectively.

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TABLE 2. Extracellular production of CPY-EGFP by A. oryzae dfc and hfm mutants

Hyphal morphology and intracellular EGFP localization.
Hyphae of dfc and hfm mutants grown in submerged cultures were observed by DIC and fluorescence microscopy (Fig. 2 and 3). For parent strain NSCE1, the hyphae were unbranched and EGFP fluorescence was observed in large spherical vacuoles that also were detected by DIC microscopy (Fig. 2A). No significant morphological abnormalities were observed for hfm-7 and hfm-21 grown at pH 5.5, except that the hyphae were slightly thicker than those of NSCE1 (Fig. 2D and E); however, dfc-14 grown at pH 5.5 had hyperbranched hyphae and diffuse EGFP fluorescence throughout the cytoplasm (Fig. 2B). hfm-4 grown at pH 5.5 also had abnormally swollen hyphae and diffuse CPY-EGFP localization (Fig. 2C). The large spherical vacuoles observed by DIC microscopy in NSCE1 were not detectable in the mutants grown at pH 5.5.

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FIG. 2. Morphological abnormality and vacuolar fragmentation in A. oryzae dfc-14 and hfm-4 mutants grown at pH 5.5. NSCE1 (A and F), dfc-14 (B and G), hfm-4 (C and H), hfm-7 (D), and hfm-21 (E) were cultivated in M medium adjusted to pH 5.5 at 30°C for 24 h. Vacuoles were stained with CMAC (F to H) and observed by differential interference contrast and fluorescence microscopy. Bars, 5 µm.

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FIG. 3. Morphological abnormality in mutants showing hyper-EGFP fluorescence in conidia. NSCE1 (A and B), hfc-3 (C and D), and hfc-4 (E and F) were cultivated in M medium adjusted to pH 5.5 (A, C, and E) or pH 8.0 (B, D, and F) at 37°C for 3 days and observed by differential interference and fluorescence microscopy. Bar, 5 µm.

To examine vacuolar morphology and to identify the compartment in which CPY-EGFP accumulated, NSCE1, dfc-14, and hfm-4 were stained with CMAC, which localizes specifically to vacuoles. Unlike the large spherical vacuoles that stained specifically with CMAC in NSCE1 (Fig. 2F), the CMAC fluorescence was dispersed throughout the cytoplasm in the two mutants (Fig. 2G and H), presumably in small vesicles. By overlaying CMAC and EGFP images, we found that the two fluorescent markers colocalized (data not shown). Thus, dfc-14 and hfm-4 grown at pH 5.5 formed abnormal hyphae, and this phenotype was associated with what appeared to be fragmented vacuoles that apparently could leak their contents.

Morphological abnormalities that were dependent on the pH of the medium also were observed in mutants with conidial hyper-EGFP fluorescence (hfc) phenotypes. hfc-3 had swollen hyphae (diameter, 15 to 20 µm) and fragmented vacuoles at pH 5.5 (Fig. 3C), even though its hyphae were as thin as those of the wild-type strain (diameter, 5 µm) and the cells contained fragmented vacuoles at pH 8.0 (Fig. 3D). In contrast, hfc-4 had unbranched hyphae and fragmented vacuoles at pH 5.5 (Fig. 3E) and hyperbranched hyphae and fragmented vacuoles at pH 8.0 (Fig. 3F).

Both dfc-14 and hfm-4 formed unbranched hyphae and large spherical vacuoles, like NSCE1, in alkaline medium (Fig. 3B and C). However, EGFP fluorescence was not observed in vacuoles but was instead seen in dots and ring-like structures around the vacuoles (Fig. 4F and G). The diameters of the dot-like structures were 0.4 to 0.6 µm, while those of the ring-like structures were 0.8 to 1.2 µm. The average number of dots in each cellular compartment was 20 for the dfc-14 and hfm-4 mutants; on average one and eight ring-like structures were present in dfc-14 and hfm-4, respectively. Overlays of fluorescence and DIC images indicated that the dot and ring-like structures colocalized with vacuoles.

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FIG. 4. Mislocalization of CPY-EGFP in A. oryzae mutants dfc-14, hfm-4, hfm-7, and hfm-21 grown at pH 8.0. NSCE1 (A), dfc-14 (B and F), hfm-4 (C and G), hfm-7 (D), and hfm-21 (E and H) were cultivated in M medium adjusted to pH 8.0 at 30°C for 24 h and observed by differential interference and fluorescence microscopy. The areas in boxes in panels B, C, and E are enlarged in panels F, G, and H, respectively. The asterisks and arrowheads indicate vacuoles and ring-like structures, respectively. The arrow in panel H indicates a ring at a hyphal tip. Bars, 5 µm.

The morphology of the hfm-7 and hfm-21 mutants grown at pH 8.0 was similar to that of the wild type; these mutants formed unbranched hyphae and large spherical vacuoles. The vacuoles lacked EGFP fluorescence (Fig. 4D and E), although the tips of the apical cells of hfm-21 did fluoresce at a position corresponding to the position of the Spitzenkörper (Fig. 4E). An enlargement showed that CPY-EGFP accumulated in a ring-like structure (Fig. 4H), which could have been the Spitzenkörper, which controls polarized filamentous growth and branching in filamentous fungi (22).

Effect of antibiotics, ambient pH, and osmotic pressure on mutant growth.
Members of one of the six complementation groups of scy mutants of A. nidulans that have increased sensitivity to cycloheximide also have a defect in vacuolar biogenesis (6). In contrast, an A. nidulans mutant lacking the avaA gene, which encodes a Rab-like GTPase, is tolerant to G418 but not to cycloheximide (15). dfc-14, hfm-4, and hfm-21, but not the wild-type parent strain, were resistant to 100 µg/ml of cycloheximide, demonstrating that some of the mutants that missort large amounts of CPY-EGFP are cycloheximide tolerant. Other changes in growth conditions, including changes in pH (pH 4 to 11), high osmotic pressure (1.2 M sorbitol and 1.2 M NaCl), and the presence of antibiotics (100 to 1,000 µg/ml G418 and 10 to 100 µg/ml nystatin) also were tested, but no obvious differences between the mutants and the wild type in terms of colony growth were observed (data not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used two different screening procedures to isolate A. oryzae vps mutants that detected either intra- or extracellular fluorescence of CPY-EGFP. Few mutants of any sort have been isolated for A. oryzae because of the difficulty of screening large populations, a problem that we overcame by using FACS to analyze a large number of conidia at one time. For most of the dfc mutants hyper-EGFP fluorescence was also released into the medium, which is consistent with our hypothesis that mutants that missort large amounts of CPY-EGFP also form conidia with dim EGFP fluorescence.

The A. oryzae mutants have some similarities to the S. cerevisiae vacuolar protein sorting mutants. The fragmented vacuoles found in dfc-14 and hfm-4 grown at pH 5.5 are similar to those found in class B vps mutants of S. cerevisiae (19), suggesting that these A. oryzae mutants have mutations in genes involved in vacuolar biogenesis. In A. nidulans, the disruption of vpsA, a homolog of S. cerevisiae VPS1 (class B), can cause fragmentation of vacuoles (26). Our results are consistent with previous observations that several VPS proteins are involved in vacuolar homotypic fusion. The dfc-14 and hfm-4 strains grown at pH 8.0 accumulated CPY-EGFP in perivacuolar dot-like structures similar to the deformed prevacuolar compartment observed in class E vps mutants of S. cerevisiae. Although more detailed analyses are necessary to identify these structures, their location and appearance are reminiscent of the class E compartment, a modified prevacuolar compartment observed in S. cerevisiae class E vps mutants (21). This similarity suggests the presence of an organelle similar to the prevacuolar compartment, which transports CPY en route to vacuoles in the vacuolar transport pathway in S. cerevisiae. If confirmed by colocalization of CPY-EGFP and homologs of prevacuolar compartment markers, this observation would be the first observation of a prevacuolar compartment in filamentous fungi.

The dfc and hfm mutants also have phenotypes specific to A. oryzae. dfc-14 and hfm-4 grown in submerged cultures at pH 5.5 have an abnormal cellular morphology that has not been reported for vps mutants of S. cerevisiae, although we must confirm that the morphological abnormality and the lack of vacuolar protein sorting result from the same mutation. We also isolated mutants with hyper-EGFP fluorescence in conidia and found that mutants that missort large amounts of CPY-EGFP tend to have abnormal hyphal morphology (Fig. 3). Therefore, at least some VPS proteins are presumed to be involved in hyphal morphogenesis in A. oryzae. In A. nidulans, a mutation in digA, a potential homolog of S. cerevisiae VPS18/PEP3, results in dichotomous and subapical branches (8), which also suggests a correlation between vacuolar protein sorting and hyphal morphogenesis. The properties of multicellularity and cell polarity specific to filamentous fungi may provide an opportunity to characterize morphological abnormalities that cannot occur in unicellular microorganisms such as S. cerevisiae. We expect that these properties will contribute to our understanding of the distinct roles of VPS proteins in filamentous fungi and yeasts. Morphological abnormalities appeared only in submerged cultures and not in solid cultures, suggesting that these strains have mutations in genes expressed in submerged cultures but not in surface cultures.

The most interesting property of A. oryzae vacuolar protein sorting mutants is the pH dependence of their phenotypes. The dfc-14 and hfm-4 mutants express their abnormal vacuolar and hyphal morphology only when they are grown at pH 5.5. In contrast, the missorting of CPY-EGFP in hfm-7 and hfm-21 was higher at pH 8.0 than at pH 5.5. Thus, the wild-type functions of the VPS proteins in A. oryzae also may be pH dependent. pH-dependent signaling proteins interact with class E VPS proteins in A. nidulans (1, 27), which could provide one mechanism by which VPS protein activity may be modulated in response to pH.

In hfm-21 grown at pH 8.0, CPY-EGFP was localized at hyphal tips in a manner similar to that observed for other extracellular enzymes, such as glucoamylase-green fluorescent protein in Aspergillus niger (9) or -amylase (13) and RNase T1-EGFP (12) in A. oryzae. Thus, we hypothesized that hfm-21 secretes CPY-EGFP via a vesicle-mediated, conventional secretory pathway similar to the one used for extracellular enzymes. Unlike glucoamylase-green fluorescent protein fluorescence (9) and RNase T1-EGFP fluorescence (12), however, CPY-EGFP fluorescence was not observed at the septa. Therefore, the vesicles transporting CPY-EGFP or -amylase may be distinguishable from those transporting glucoamylase or RNase T1 in this mutant. In an enlargement, CPY-EGFP appeared to accumulate in a ring-like structure, at a position that corresponded to that of the Spitzenkörper, which controls polarized filamentous growth and branching in filamentous fungi (22). These structures, which were not observed when other extracellular enzyme markers in filamentous fungi were used, have previously been seen only with FM4-64, a marker of endocytosis (7). These results suggest that CPY-EGFP may be transported by a novel pathway possibly involving the Spitzenkörper.

Confirmation that the mutant phenotypes are the result of single gene mutations requires identification of a specific lesion in each mutant. We are now trying to identify the mutations in the dfc and hfm mutants by complementation with transformed genomic library clones of wild-type A. oryzae. Since dfc and hfm mutants have phenotypes specific to A. oryzae, as well as characteristics that are more widespread, we expect that our set of mutants contains not only mutants with S. cerevisiae VPS homologues but also mutants with mutations in A. oryzae-specific genes. Identification of VPS genes should enhance molecular biological analyses and contribute to elucidation of vacuolar protein sorting mechanisms in A. oryzae. The mutants also may be useful in elucidating cellular mechanisms underlying morphogenesis, organelle biogenesis, protein sorting, pH response, and tolerance to antibiotics, all of which rely on correct vacuolar function.

 
This study was supported by grant-in-aid for scientific research (B) 15380058 to K. Kitamoto from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a program for the promotion of basic research activities for innovative biosciences of the Bio-Oriented Technology Research Advancement Institution.

 
* Corresponding author. Mailing address: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81-3-5841-5161. Fax: 81-3-5841-8033. E-mail: akitamo{at}mail.ecc.u-tokyo.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, August 2005, p. 4856-4861, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4856-4861.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Shoji, J.-y., Arioka, M., Kitamoto, K. (2006). Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae. Eukaryot Cell 5: 411-421 [Abstract] [Full Text]  

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