Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae

Abstract

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra-and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both ⌬vam3 and ⌬pep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.Vacuoles are acidic compartments that are important for metabolite storage and cytosolic ion and pH homeostasis (for a review, see reference 18). In the unicellular yeast Saccharomyces cerevisiae, roles of and transport pathways to vacuoles have been extensively studied. Especially, many studies have been focused on the syntaxin family t-SNAREs (target organelle soluble N-ethyl-maleimide-sensitive factor [NSF] attachment protein receptors), which are central molecules of intracellular vesicular transport. t-SNAREs that reside on the target organelle membrane mediate the fusion of transport vesicles with the organelles via the specific interaction with vesicle SNAREs on the transport vesicles (29). S. cerevisiae Pep12p is the syntaxin family t-SNARE on the late endosome/ prevacuolar compartment and receives Golgi-derived vesicles (3). Vam3p is the vacuolar membrane syntaxin that regulates the vesicular traffic to and the homotypic fusion of vacuoles (44). Since S. cerevisiae Vam3p localizes exclusively on the vacuolar membrane, Vam3p is frequently used for visualization of vacuoles (45, 46) and as a marker of vacuoles in subcellular fractionation experiments. AtVam3p is a Vam3p-homologue protein in Arabidopsis thaliana (30), and its fusion protein with...

Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

“…In the region before the first septum the vacuolar markers were abundant in an interconnected network of thin (Ͻ1 m) tubules, often with small, attached vesicles. These results with N. crassa are generally consistent with observations made using GFPtagged VAM-3 in Aspergillus oryzae (45,54). N. crassa grows faster, and the diameter of hyphae can be fivefold larger.…”

“…GFP and fluorescent dyes have been used successfully to examine nuclei and mitochondria (24,26). Several reports have shown that "the vacuole" is far more dynamic and complex than the textbook presentation of a large spherical organelle (10,24,31,33,54). Our understanding of the structure and abundance of the ER and the Golgi body is limited.…”

Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans*

“…We examined vacuolar morphology in the V o a mutants by staining live cells with FM4-64 and CMAC. Upon endocytosis, the lipophilic dye FM4-64 stains vacuolar membranes (46), whereas CMAC passively accumulates in the vacuolar lumen (47). Fig.…”

Vsl1p cooperates with Fsv1p for vacuolar protein transport and homotypic fusion in Schizosaccharomyces pombe

“…This functional relationship can also be seen in the filamentous fungus Aspergillus oryzae, where AoVam3p is the sole orthologue of both Sac. cerevisiae Vam3p and Pep12p (Shoji et al, 2006;Kuratsu et al, 2007). Furthermore, although Vsl1p is thought to be the counterpart of Vam7p as we elucidated here, Vsl1p has functional redundancy with Fsv1p.…”