title: Characterization of a Novel Prevacuolar Compartment in Neurospora crassa.
abstract: Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California, USA bbowman@ucsc.edu.
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Bowman
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California, USA.
authors.forename: Marija
authors.initials: M
authors.lastname: Draskovic
authors.affiliation: School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Schnittker
authors.affiliation: School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA.
authors.forename: Tarik
authors.initials: T
authors.lastname: El-Mellouki
authors.affiliation: School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Plamann
authors.affiliation: Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California, Mexico.
authors.forename: Eddy
authors.initials: E
authors.lastname: Sánchez-León
authors.affiliation: Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California, Mexico.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Riquelme
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California, USA.
authors.forename: Emma Jean
authors.initials: EJ
authors.lastname: Bowman
mesh_terms: D000225:Adenine / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects; D000072159:Dynactin Complex; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D025301:Hyphae / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009492:Neurospora crassa / Q000187:drug effects / Q000378:metabolism*; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism; D010860:Pigments, Biological / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000072159:Dynactin Complex; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D010860:Pigments, Biological; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; D020691:rab GTP-Binding Proteins; D000225:Adenine