Structural and Functional Properties of Plasma Membranes from the Filamentous Fungus <i>Penicillium chrysogenum</i>

Hillenga, Dirk J.; Versantvoort, Hanneke J.M.; Driessen, Arnold J. M.; Konings, Wil N.

doi:10.1111/j.1432-1033.1994.t01-1-00581.x

Cited by 19 publications

(21 citation statements)

References 30 publications

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“…P. chrysogenum cells were grown on (1) YGG medium containing 0.8% KCl, 1.6% glucose, 0.67% yeast nitrogen base (Difco), 0.15% citric acid, 0.6% K 2 HPO 4 , 0.2% yeast extract pH 6.2 supplemented with penicillin and streptomycin (Gibco) or (2) penicillin production medium (Hillenga et al 1994) supplemented with 0.05% phenoxyacetic acid. P. chrysogenum transformants were selected on media with acetamide as sole nitrogen source as described (Cantoral et al 1987).…”

Section: Strains and Mediamentioning

confidence: 99%

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

Kiel

Berg

Fusetti

et al. 2009

Funct Integr Genomics

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In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.

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Section: Strains and Mediamentioning

confidence: 99%

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

Kiel

Berg

Fusetti

et al. 2009

Funct Integr Genomics

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“…To study transport processes that play a role in penicillin biosynthesis, a procedure was developed for the isolation of plasma membranes from P. chrysogenum (7). Hybrid membrane vesicles were obtained by fusing plasma membranes with cytochrome c oxidase vesicles.…”

Section: Discussionmentioning

confidence: 99%

“…P. chrysogenum plasma membranes were isolated by the procedure described by Hillenga et al (7). Cytochrome c oxidase-containing liposomes (10 mg of lipid) and plasma membranes (1 mg of protein) were mixed, rapidly frozen in liquid N 2 , and thawed slowly at 21ЊC.…”

Section: Methodsmentioning

confidence: 99%

Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum

et al. 1996

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The characteristics of the basic amino acid permease (system VI) of the filamentous fungus Penicillium chrysogenum were studied in plasma membranes fused with liposomes containing the beef heart mitochondrial cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes accumulated the basic amino acids arginine and lysine. Inhibition studies with analogs revealed a narrow substrate specificity. Within the external pH range of 5.5 to 7.5, the transmembrane electrical potential (⌬) functions as the main driving force for uphill transport of arginine, although a low level of uptake was observed when only a transmembrane pH gradient was present. It is concluded that the basic amino acid permease is a H ؉ symporter. Quantitative analysis of the steady-state levels of arginine uptake in relation to the proton motive force suggests a H ؉ -arginine symport stoichiometry of one to one. Efflux studies demonstrated that the basic amino acid permease functions in a reversible manner.Amino acids are utilized by fungi as primary or secondary nitrogen sources or as building blocks for the synthesis of proteins and peptides. Systems involved in the translocation of amino acids across the plasma membrane have been studied in only a few filamentous fungi (5,8,25). Of these fungi, Neurospora crassa, Aspergillus nidulans, and, to a lesser extent, Penicillium chrysogenum are genetically and biochemically the most extensively characterized species. Two distinct classes of plasma membrane-located amino acid permeases are found in filamentous fungi: systems that catalyze the uptake of structurally related amino acids with a broad substrate specificity, such as the general amino acid permeases of plant and animal cells (2,15,18), and systems with a narrow substrate specificity, such as bacterial amino acid transporters (22).Fungi show a peculiar substrate specificity in their amino acid transport systems, and multiple transport mechanisms seem to exist for several amino acids. In N. crassa, five distinct transport systems have been identified, with specificity for aromatic and aliphatic amino acids (system I); aromatic, aliphatic, and basic amino acids (system II); basic amino acids (system III); acidic amino acids (system IV); and L-methionine (system V) (5,8,20,25). Studies with mycelium of P. chrysogenum indicate that this fungus possesses at least six distinct amino acid transport systems (1, 5, 8-10, 11, 24). Most of these systems are specific for one amino acid and analogs, except for system III, which is a general amino acid permease, and system IV, which transports acidic amino acids only (8, 10). Amino acid transporters of fungi are assumed to possess some typical properties: (i) they seem to function unidirectionally; i.e., only uphill transport is observed, while efflux or countertransport of the accumulated amino acids is not detected (8, 19); and (ii) their activity is regulated by transinhibition, i.e., a high internal concentration of an amino acid appears to lower the activity of the transport syst...

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“…The complex mycelial morphology and the presence of intracellular compartments make it impossible to relate the uptake of sulfate in a quantitative manner to the driving forces. We therefore reinvestigated the mechanism of sulfate uptake in P. chrysogenum using a well-defined model system, i.e., isolated plasma membranes fused with cytochrome c oxidase-containing liposomes (5).…”

mentioning

confidence: 99%

“…P. chrysogenum Wisconsin 54-1255 (kindly supplied by Gist-brocades NV) was first grown for 70 h at 25ЊC on production medium (pH 6.3) (10) supplemented with 10 mM glutamate and 10% (mass/vol) glucose and subsequently incubated for 16 h in production medium in which all sulfate salts were replaced by equivalent chloride salts. Plasma membranes were isolated by a method previously described (5) and fused with cytochrome c oxidase-containing liposomes (3, 4) composed of 61% (by mass) acetone-ether-washed Escherichia coli lipid, 20% (by mass) egg yolk L-phosphatidylcholine, and 19% (by mass) ergosterol (3,5). Fusion was induced by a repeated freeze-thawing step, and the membranes were sized by extrusion through polycarbonate filters (Avestin Inc., Ottawa, Canada) with pore diameters of 400 and 200 nm (5).…”

mentioning

confidence: 99%

Sulfate transport in Penicillium chrysogenum plasma membranes

et al. 1996

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Transport studies with Penicillium chrysogenum plasma membranes fused with cytochrome c oxidase liposomes demonstrate that sulfate uptake is driven by the transmembrane pH gradient and not by the transmembrane electrical potential. Ca 2؉ and other divalent cations are not required. It is concluded that the sulfate transport system catalyzes the symport of two protons with one sulfate anion.One of the transport processes which plays an important role in the biosynthesis of penicillins by filamentous fungi is the uptake of sulfate. Current penicillin production processes yield final penicillin titers which are more than 500 times higher than those obtained with the parental Penicillium chrysogenum strain NRRL 1951 (9). Sulfate is the primary sulfur source in industrial production of penicillin and is the precursor of cysteine that together with valine forms the backbone of the penicillin molecule (9, 15). Therefore, the strongly increased demand for cysteine during penicillin production requires an elevated rate of sulfate uptake from the environment. Studies of the mechanism of sulfate uptake by P. chrysogenum, related filamentous fungi, and yeasts have led to the conclusion that this process occurs through active transport (1, 6, 8, 12-14, 16, 17). It has been suggested that in Penicillium notatum, sulfate is taken up in symport with one proton and one calcium ion (2). According to such a mechanism, the driving forces for sulfate uptake would be the transmembrane electrical potential (⌬⌿), the transmembrane pH gradient (⌬pH), and the chemical gradient of calcium ions (⌬ Ca 2ϩ/F). Although sulfate transport was shown to promote uptake of Ca 2ϩ , a stoichiometric coupling could not be demonstrated. It has been suggested that the sulfate transport system exchanges SO 4. Since these studies have been performed with metabolically active mycelium, the possibility of involvement of plasma membrane-localized Ca 2ϩ transport systems and Ca 2ϩ ATPases cannot be excluded. Moreover, sulfate appears to be sequestered in two distinct intracellular pools, one of which only slowly exchanges with external sulfate (7). The complex mycelial morphology and the presence of intracellular compartments make it impossible to relate the uptake of sulfate in a quantitative manner to the driving forces. We therefore reinvestigated the mechanism of sulfate uptake in P. chrysogenum using a well-defined model system, i.e., isolated plasma membranes fused with cytochrome c oxidase-containing liposomes (5).Sulfate transport in fused plasma membranes. P. chrysogenum shows a high level of sulfate uptake activity when cells are deprived of sulfur (19). P. chrysogenum Wisconsin 54-1255 (kindly supplied by Gist-brocades NV) was first grown for 70 h at 25ЊC on production medium (pH 6.3) (10) supplemented with 10 mM glutamate and 10% (mass/vol) glucose and subsequently incubated for 16 h in production medium in which all sulfate salts were replaced by equivalent chloride salts. Plasma membranes were isolated by a method previously described (5) and f...

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Structural and Functional Properties of Plasma Membranes from the Filamentous Fungus Penicillium chrysogenum

Cited by 19 publications

References 30 publications

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum

Sulfate transport in Penicillium chrysogenum plasma membranes

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