Two Fluorescent Markers Identify the Vacuolar System of Schizophyllum commune Amy L. Inselman,*Allen C. Gathman, Walt W. Lilly Department of Biology, Southeast Missouri State University, Cape Girardeau, MO 63701, USA Received: 23 December 1998 / Accepted: 11 January 1999 Abstract. Vacuole-mediated proteolysis is important to sustained growth of filamentous wood-decaying fungi such as Schizophyllum commune. Demonstrating that specific proteases are vacuole associated has been difficult in these organisms due to the lack of specific markers for vacuolar compartments. We used 5-(and 6-)-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) and a proprietary vacuolar membrane marker for yeast (MDY-64; Molecular Probes) for in situ fluorescent labeling of the vacuoles of S. commune mycelia grown on microscope slides. MDY-64 labels numerous small vesicles in S. commune mycelia in addition to larger vacuolar structures. In contrast, carboxy-DCFDA apparently is taken up by a subset of the MDY-64-labeled vesicles, accumulating primarily in larger vacuoles. Staining of mycelia with carboxy-DCFDA shows a transition from mostly cytoplasmic fluorescence in apical cells with little vacuolar fluorescence to nearly complete sequestration of the stain in vacuoles of older cells. In penultimate cells, both cytoplasm and vacuolar structures fluoresce. Vacuoles stained with carboxy- DCFDA typically were spherical and ranged in size from 0.4 μm to 3.2 μm in diameter with a mean of 1.8 um. Occasionally, in penultimate cells, tubular structures which stained with carboxy-DCFDA were found. ScPrB, a principal enzyme of nitrogen-limitation induced autolysis in S. commune, copurified in sucrose density gradients with carboxy-DCFDA and acid phosphatase, demonstrating its vacuolar localization. The wood-decaying basidiomycete Schizophyllum com- mune is a model system for the study of nitrogen- limitation induced autolysis in filamentous fungi. In S. commune, transfer of exponentially growing mycelium to nitrogen-poor medium results in activation of proteolytic enzyme activity [22], proteolytic release of incorporated amino acids, and their subsequent transfer from older mycelia to the growing mycelial margin [17]. If S. commune is like Saccharomyces cerevisiae, the principal model system in which fungal autolysis has been studied, the vacuole and its associated proteolytic enzymes are integrally involved [7, 24]. In S. commune, the metallopro- tease ScPrB appears to be one of the major proteases involved in nitrogen limitation–induced autolysis [8]. This protease is intracellular [12], and it may be localized to vacuoles [25; Lilly unpublished data]. Traditionally regarded as analogous to the central vacuoles of higher plant cells, the vacuoles of fungal cells are important organelles for turgor regulation, storage, and lytic activity [13]. Techniques that allow the isolation of subcellular fractions enriched in vacuoles from S. cerevisiae [21] and Neurospora crassa [3] have been particularly important in defining the storage [6] and lytic [24] roles of these structures. Unfortunately, these meth- ods also yield populations of homogeneous vacuolar/ vesicular structures, contributing to the widely held view of vacuoles as morphologically and biochemically uni- form organelles. Recent advances in microscopic tech- niques, such as transmission electron microscopy of freeze-substituted hyphae and the use of vacuole-specific fluorescent markers, have revealed a far more complex structure for the vacuolar system of filamentous fungi [2]. These studies have indicated that the vacuolar system comprises a reticulated network of tubules, vesicles, and larger spherical organelles [1, 10, 23]. This system is dynamic, and arguments have been made for its analogy, *Present address: Program in Biochemistry/Molecular and Cellular Biology, University of Tennessee, Knoxville, TN, USA Correspondence to: W.W. Lilly CURRENT MICROBIOLOGY Vol. 38 (1999), pp. 295–299 An International Journal  Springer-Verlag New York Inc. 1999