Methods for mycelial breakage and isolation of mitochondria and vacuoles of neurospora

Cramer, Carole L.; Ristow, Janet L.; Paulus, Thomas J.; Davis, Rowland H.

doi:10.1016/0003-2697(83)90390-1

Cited by 61 publications

(34 citation statements)

References 12 publications

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“…To determine where calcium is located within hyphae, we used a fractionation procedure developed to isolate mitochondria, vacuoles, and lighter membranes (Fig. 3) (7,13,40). We grew wild-type mycelium in bubbling cultures with vigorous aeration in Vogel's medium N containing 45 Ca (0.68 mM CaCl 2 ), broke the hyphae with glass beads, separated membrane fractions by differential centrifugation, and estimated the amount of calcium in each fraction by counting 45 Ca.…”

Section: Resultsmentioning

confidence: 99%

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Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

et al. 2011

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We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca 2؉ -ATPase, or NCA-3, a PMC-type Ca 2؉ -ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca 2؉ -ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4-to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca 2؉ /H ؉ exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that "the vacuole" is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.

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Section: Resultsmentioning

confidence: 99%

“…To collect cell fractions, we used a well-characterized procedure developed to isolate mitochondria, vacuoles, and other organelles from N. crassa (7,13,40). Strains were grown in 1 or 2 liters of Vogel's medium containing approximately 50 Ci of 45 Ca.…”

Section: Methodsmentioning

confidence: 99%

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

et al. 2011

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“…Vacuoles prepared from 40 h mycelium of P. notaturn, by the method of Cramer et al (1983), were numerous but of small size (< 100 nm). A larger range of sizes (0.1-1.0 pm) was obtained from both vegetative and calcium-induced mycelium, using the method of Cornelius et al (1989) and showed high marker H+-ATPase activity.…”

Section: Methodsmentioning

confidence: 99%

Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Penicillium notatum

Pitt

Barnes²

1993

Journal of General Microbiology

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~ ~~~~~~Cytosolic free calcium concentration [Ca2+], of protoplasts from Penicillium notatum was measured using the permeant acetoxy ester (quin-2-AM) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5.8-7.0 and its subsequent hydrolysis was low. Uptake was promoted by an external pH of 5.0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7.2, which was near the optimum of the carboxylic esterases responsible for the hydrolysis. Uptake of Ca2+ was biphasic with the average cell calcium concentration of protoplasts increasing from an initial value of 2 pmol to 50 pmol (kg cell water)-', during attainment of the steady state after 30 min, at which time [Ca2+], was unchanged at 20 nM but increased to 182 nM at 2-6 h exposure to 2.5 mM-Ca2+. Broadly similar changes in [Ca2+], were found in protoplasts derived from mycelium samples exposed to Ca2+ over the same period of time. The location of Ca2+ was determined in subfractionated organelles and characterized using enzyme markers and electron microscopy. In 32 h mycelium preloaded with Ca2+ for 6 h, Ca2+ was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacuoles and plasma membrane components. Calcium was not released by inositol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subcellular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the addition of 2-5 p~-A23187. The anti-calmodulin agent calmidazolium (R24571) greatly reduced sporulation when addition preceded that of Ca2+. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571, whilst 1 h and 4-6 h protein phosphorylations, which were also present to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca2+-induced mycelium had enhanced capacity for calmodulin-mediated phosphorylation relative to corresponding vegetative cells and that complex differential changes in such phosphorylations occurred during Ca2+-induction of the sporulation process.

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“…The cells (ca. 100 g [wet weight]) were lysed in isolation buffer with a bead beater in lots of 25 g (15). A crude extract was obtained after removing cell debris by centrifugation (2,000 x g for 10 min) and filtration through Whatman 934AH glass fiber filters.…”

Section: Methodsmentioning

confidence: 99%

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

Chow¹,

Metzenberg²,

RajBhandary³

1989

Mol. Cell. Biol.

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We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the keu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by Si nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the ku-S mutation had been mapped before. We show that the ku-S strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the ku-S strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in ku-S extracts, the ku-S strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.The mitochondria of eucaryotic cells possess a DNA genome which is distinct from that in the nucleus. The mitochondrial DNA is replicated, transcribed, and used to synthesize proteins within the mitochondrial matrix. However, the coding capacity of most mitochondrial genomes is limited to the tRNAs and rRNAs required for protein synthesis and the mRNAs for a few proteins, most of which are involved in electron transport (8). The mitochondria are almost completely dependent upon the nuclear genome for the proteins that function within them. These nucleusencoded proteins are synthesized in the cytoplasm and imported into the mitochondrion. Thus, the nuclear genome encodes proteins which are required for analogous functions in the mitochondria and the cytoplasm (protein synthesis and energy metabolism) and in the mitochondria and the nucleus (DNA replication, RNA synthesis, most tRNA modifications, etc.).Work with the yeast Saccharomyces cerevisiae has suggested two strategies for specifying differential localization of proteins performing the same basic functions. For histidyl-tRNA synthetase (43), valyl-tRNA synthetase (10), and several of the tRNA-modifying enzymes (18,20,29), differential transcription of single genes with alternate in-frame protein synthesis initiation sites allows for two forms to be made and localized to the appropriate compartment. The 5' end of the mRNA for these enzymes determines which form of the enzyme...

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Methods for mycelial breakage and isolation of mitochondria and vacuoles of neurospora

Cited by 61 publications

References 12 publications

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Penicillium notatum

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

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