Cloning and characterization of a gene (avaA) from Aspergillus nidulans encoding a small GTPase involved in vacuolar biogenesis

Ohsumi, Keisuke; Arioka, Manabu; Nakajima, Harushi; Kitamoto, Katsuhiko

doi:10.1016/s0378-1119(02)00626-1

Cited by 30 publications

(35 citation statements)

References 19 publications

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“…3A) that mediates fusion of vesicles and other prevacuolar compartments to vacuoles (18). We disrupted A. parasiticus vb1 that encodes a homolog of Ypt7 (one member of the tethering complex) in yeast and AvaA in Aspergillus nidulans that are necessary for vacuole biogenesis; ypt7 and avaA disruption results in fragmented vacuoles (19,20). Vb1 contains 205 aa and is 70% identical to yeast Ypt7, 74% identical to mammalian Rab7 GTPases, and 94% identical to A. nidulans AvaA.…”

Section: Blocking Vesicle-vacuole Fusion Increases Aflatoxin Synthesimentioning

confidence: 99%

A key role for vesicles in fungal secondary metabolism

Chanda

Roze

Kang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

196

244

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Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B 1 ; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.S econdary metabolites, natural products generated by filamentous fungi, plants, bacteria, algae, and animals, have an enormous impact on humans due to their application in health, medicine, and agriculture. Many secondary metabolites are beneficial (antibiotics, statins, morphine, etc.), though phytotoxins (e.g., ricin, crotin, amygdalin) and fungal poisons called mycotoxins (e.g., aflatoxin, sterigmatocystin, fumonisin) are detrimental to humans and animals. To control or customize biosynthesis of these natural products we must understand how and where secondary metabolism is orchestrated within the cell.Vacuoles and vesicles are known to sequester secondary metabolites to protect host cells from self-toxicity (1). Enzymes involved in secondary metabolism are often found in vesicles and vacuoles, including those for biosynthesis of alkaloids (e.g., berberine, sanguinarine, camptotecin, and morphine; reviewed in refs. 1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the ␤-lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6-8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of...

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Section: Blocking Vesicle-vacuole Fusion Increases Aflatoxin Synthesimentioning

confidence: 99%

A key role for vesicles in fungal secondary metabolism

Chanda

Roze

Kang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

196

244

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“…VAM4/YPT7 encodes a S. cerevisiae gene that is required for vacuole fusion (Wichmann et al, 1992). Deletion of the VAM4/YPT7 homologue avaA in A. nidulans, however, was shown not to reduce the hyphal growth rate, although this mutant exhibited highly fragmented vacuoles (Ohsumi et al, 2002).…”

Section: Growth Defects Of the A Gossypii Wal1 Mutantmentioning

confidence: 99%

Apical localization of actin patches and vacuolar dynamics in Ashbya gossypii depend on the WASP homolog Wal1p

Walther

Wendland

2004

Journal of Cell Science

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Analysis of the Ashbya gossypii Wiskott-Aldrich syndrome-like gene AgWAL1 indicates that it is required for the maintenance of polarized hyphal growth. Growth and organelle dynamics of the wild type and of wal1 and other mutant strains were monitored by in vivo (fluorescence) time-lapse microscopy. Loss of WAL1 led to slow growth and defects in polarized growth that produced swellings in subapical regions, whereas formation of hyphal tips and dichotomous tip branching occurred as in the wild-type. Few actin cables in Agwal1 cells were found to insert into the hyphal tip, but specific clustering of cortical actin patches was observed in subapical regions of hyphal tips instead of at the hyphal apex. Distribution and movement of vacuoles was observed in vivo using FM4-64. In the wild type and in the slowly growing mutant strains bem2 and cla4, which lack a Rho-GTPase-activating protein and a PAK kinase, respectively, early endosomes appeared in the hyphal tip, whereas very few early endosomes and small vacuoles were found in the wal1 mutant hyphal tips, thus linking the cortical patch defect of wal1 hyphae with the distribution of endosomes. Vivid movement of vacuoles seen in the wild type and in the bem2 mutant in subapical regions was largely reduced in the wal1 and cla4 mutants. The tubular structure of mitochondria (as visualized by DIOC6 in vivo) was similar in the wild type and the wal1 mutant, although wal1 mitochondria appeared to be larger. Interestingly, mitochondria were found to insert into the hyphal tips in both strains. Our results indicate a function for Wal1p in filamentous fungi in coordinating actin patch distribution with polarized hyphal tip growth.

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“…Rab7, which is a gene identified in Homo sapiens, controls the late stages of endocytosis, including early to late endosomal fusion and transport between late endosomes and lysosomes (6). It also plays a role in vacuolar biogenesis and the targeting of proteins to the vacuole (42). Rab7 appears to be conserved across species, and many of its homologues have been found in both plants (22) and fungi (42,66).…”

mentioning

confidence: 99%

“…It also plays a role in vacuolar biogenesis and the targeting of proteins to the vacuole (42). Rab7 appears to be conserved across species, and many of its homologues have been found in both plants (22) and fungi (42,66). Recently, several interacting partners of Rab7 have been isolated, such as RILP (for Rab-interacting lysosomal protein) (7), Rabring 7 (36), and ORP-1L [for oxysterol-binding protein-related protein-1L] (25).…”

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confidence: 99%

Endocytosis in the Shiitake Mushroom Lentinula edodes and Involvement of GTPase LeRAB7

Lee¹,

Szeto

Ng³

et al. 2007

Eukaryot Cell

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Endocytosis is the process by which substrates enter a cell without passing through the plasma membrane but rather invaginate the cell membrane and form intracellular vesicles. Rab7 regulates endocytic trafficking between early and late endosomes and between late endosomes and lysosomes. LeRab7 in Lentinula edodes is strongly homologous to Rab7 in Homo sapiens. Receptors for activated C kinase-1 (LeRACK1) and Rab5 GTPase (LeRAB5) were isolated as interacting partners of LeRab7, and the interactions were confirmed by in vivo and in vitro protein interaction assays. The three genes showed differential expression in the various developmental stages of the mushroom. In situ hybridization showed that the three transcripts were localized in regions of active growth, such as the outer region of trama cells, and the subhymenium of the hymenophore of mature fruiting bodies and the prehymenophore of young fruiting bodies. The existence of endocytosis in the mycelium and hymenophores was confirmed by the internalization of FM4-64. LeRAB7 was partially colocalized with the AM4-64 and was located in the late endocytic pathway. This is the first report of the presence of endocytosis in homobasidiomycetes. LeRAB7, LeRAB5, and LeRACK1 may contribute to the growth of L. edodes and cell differentiation in hymenophores.

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Cloning and characterization of a gene (avaA) from Aspergillus nidulans encoding a small GTPase involved in vacuolar biogenesis

Cited by 30 publications

References 19 publications

A key role for vesicles in fungal secondary metabolism

A key role for vesicles in fungal secondary metabolism

Apical localization of actin patches and vacuolar dynamics in Ashbya gossypii depend on the WASP homolog Wal1p

Endocytosis in the Shiitake Mushroom Lentinula edodes and Involvement of GTPase LeRAB7

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