A Study of the Protein Secretory Pathway of Aspergillus niger Using a Glucoamylase–GFP Fusion Protein

Khalaj, Vahid; Brookman, Jayne L.; Robson, Geoffrey D.

doi:10.1006/fgbi.2000.1245

Cited by 35 publications

(24 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Within 25 min of applying BFA, both the ST-RFP and CopA-GFP patterns became less distinct, as expected, since BFA dissociates COPI from vesicles and later affects GE morphology by perturbing vesicle trafficking (Jackson & Casanova, 2000). Khalaj et al (2001) showed that 100 mg BFA ml 21 induces perinuclear endomembrane redistributions in Aspergillus niger after 2 h. Our milder and shorter BFA treatments had less extreme effects, but were consistent with more dramatic redistributions over longer timescales.…”

Section: Discussionsupporting

confidence: 81%

See 1 more Smart Citation

Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials

Hubbard

Kaminskyj

2008

Microbiology

View full text Add to dashboard Cite

Golgi equivalents (GEs) process materials in the fungal secretory pathway. Despite the importance of localized secretion in fungal tip growth, GE behaviour in living hyphae has not been documented. The distribution was monitored of an Aspergillus nidulans putative GE-associated protein, CopA, tagged with GFP (CopA-GFP). This co-localized with a Golgi body/GE marker established in other systems, a-2,6-sialyltransferase, tagged with red fluorescent protein (ST-RFP). CopA-GFP and ST-RFP distributions responded similarly to brefeldin A, which impairs Golgi/GE trafficking. We used a CopA-GFP, hypA1 strain to study GE distribution and behaviour in growing A. nidulans hyphae. This strain has a wild-type phenotype at 28 6C, can be manipulated by changing growth temperature or by use of cytoskeleton inhibitors, and its GE behaviour is consistent with that in a wild-type-morphology strain. A. nidulans GEs were more abundant at hyphal tips than subapically, and showed saltatory motility in all directions. Anterograde GE movements predominated. These were positively correlated with, but at least 10-fold faster than, hyphal growth rate, under all growth and experimental conditions investigated. The actin inhibitor latrunculin B reduced both anterograde GE movement and hyphal growth rate, whereas the microtubule (MT) depolymerizer benomyl increased anterograde GE movement and decreased hyphal growth rate. The MT stabilizer taxol increased A. nidulans GE movement but not hyphal growth rate. A. nidulans GE motility appears to have a complex dependence on both actin and MTs. We present a model for apical delivery of growth materials in which A. nidulans GEs play a role in long-distance transport. INTRODUCTIONFungal tip growth uses the coordinated activities of the endomembrane and cytoskeletal systems to target growth materials to the hyphal apex (Bartnicki-Garcia, 2002;Heath, 1990Heath, , 1995. Golgi bodies process and sort materials (Farquhar & Palade, 1981, 1998Matheson et al., 2006;Mogelsvang & Howell, 2006), including those destined for secretion. Fungal Golgi equivalents (GEs) differ morphologically from Golgi bodies in animals and plants but perform similar functions (Beckett et al., 1974;Bentivoglio & Mazzarello, 1998;Cole et al., 2000), making them central players in the tip growth process. As in most fungi, Aspergillus nidulans GEs imaged with transmission electron microscopy (TEM) have single pleiomorphic cisternae (Beckett et al., 1974;Kaminskyj & Boire, 2004; Kurtz et al., 1994).The distribution of organelles (Bartnicki-Garcia, 2002) and proteins (McGoldrick et al., 1995;Sharpless & Harris, 2002) can be used to infer their function. Evidence from A. nidulans (Breakspear et al., 2007), Aspergillus oryzae (Akao et al., 2006), Candida albicans (Rida et al., 2006) and Saccharomyces cerevisiae (Matsuura-Tokita et al., 2006) suggests that GE distribution in fungi is related to tip growth.The behaviour of organelles in living A. nidulans hyphae has been revolutionized by fluorescent protein tagging (e.g. Suelmann & Fischer...

show abstract

Section: Discussionsupporting

confidence: 81%

“…(Jackson & Casanova, 2000). Khalaj et al (2001) showed that 100 mg BFA ml 21 induces perinuclear endomembrane redistributions in Aspergillus niger after 2 h. Our milder and shorter BFA treatments had less extreme effects, but were consistent with more dramatic redistributions over longer timescales. …”

supporting

confidence: 61%

Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials

Hubbard

Kaminskyj

2008

Microbiology

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Discussionsupporting

confidence: 65%

Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

Ohneda

Arioka

Kitamoto

2005

Appl Environ Microbiol

View full text Add to dashboard Cite

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.

show abstract

“…In the wild-type strain, an intense signal corresponding to AmyB-EGFP was detected at the hyphal tip (Fig. 6A), similar to the localization of the Spitzenkörper, the location where exocytotic secretory components and secretory proteins concentrate in filamentous fungi (23,(36)(37)(38). Under overexpression conditions, mDsRed-AoVip36 was detected as punctuate structures, whereas the intracellular AmyB-EGFP signal seemed to be decreased and the localization was more prominent at the cell periphery (Fig.…”

Section: Resultsmentioning

confidence: 52%

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Hoang

Maruyama

Kitamoto

2015

Appl Environ Microbiol

View full text Add to dashboard Cite

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme ␣-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of ␣-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. Filamentous fungi possess a prominent secretory capacity and, thus, are excellent hosts for recombinant protein production. Numerous approaches and attempts have been made to produce industrially valuable proteins in filamentous fungi, such as Aspergillus and Trichoderma (1). However, higher eukaryotic proteins are generally inefficiently produced and secreted in these fungi compared to the production and secretion of fungal or endogenous proteins. Several bottlenecks in the heterologous protein production process have been identified to date, and a few limiting factors have been overcome by genetically modifying the expression host. In particular, reducing protease activity is necessary to limit the degradation of heterologous proteins, as was demonstrated by the 3-fold increase in the level of heterologous proteins in the culture supernatant of an Aspergillus oryzae strain with 10 protease genes deleted (2). Heterologous protein production by A. oryzae was also effectively improved by the repression of vacuolar protein sorting and autophagy (3, 4). The genetic fusion of a target protein with an endogenous protein carrier is a commonly used strategy to increase he...

show abstract

A Study of the Protein Secretory Pathway of Aspergillus niger Using a Glucoamylase–GFP Fusion Protein

Cited by 35 publications

References 63 publications

Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials

Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials

Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Contact Info

Product

Resources

About