Candida maltosa NADPH‐cytochrome P450 Reductase: Cloning of a Full‐length cDNA, Heterologous Expression in Saccharomyces cerevisiae and Function of the N‐terminal Region for Membrane Anchoring and Proliferation of the Endoplasmic Reticulum

Kärgel, Eva; Menzel, Ralph; Honeck, Horst; Vogel, Frank; Böhmer, Annette; Schunck, Wolf‐Hagen

doi:10.1002/(sici)1097-0061(19960330)12:4<333::aid-yea915>3.3.co;2-3

Cited by 25 publications

(29 citation statements)

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“…CPR was shown to face the cytosol 15,16 and many cytochrome p450s were found on ER membranes facing the cytosol as well. This suggested that HO1 would have to face the cytosol to have direct access to its electron donor CPR.…”

Section: Resultsmentioning

confidence: 99%

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol

et al. 2012

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Section: Resultsmentioning

confidence: 99%

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol

et al. 2012

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“…Antibodies used for immunofluorescence microscopy, preparation of cells by formaldehyde fixation, and staining of cells with DAPI (4Ј,6Ј-diamidino-2-phenylindole) were described before (42,47). Immunoelectron microscopy was carried out as described previously (22,53). Briefly, yeast cells were harvested by centrifugation and fixed for 1 h with 4% formaldehyde and 0.5% glutaraldehyde under culture conditions (pH 5.5, 30°C), cryoprotected by a mixture of 25% polyvinylpyrrolidone (PVP K15; molecular weight, 10,000; Fluka) and 1.6 M sucrose (50) for 3 h, and frozen in liquid nitrogen.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Nup2p, a Yeast Nucleoporin, Functions in Bidirectional Transport of Importin α

Solsbacher

Maurer

Vogel

et al. 2000

Molecular and Cellular Biology

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124

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Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin ␣ and importin ␤. Srp1p, the Saccharomyces cerevisiae homologue of importin ␣, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin ␣-importin and ␤ and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin ␤) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin ␣-importin ␤ complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.Protein transport across the nuclear envelope is mediated by soluble receptors of the importin ␤ family. The receptors interact with their cargo either directly or via an adapter molecule (for reviews, see references 13 and 30). Nuclear import and nuclear export signals within the proteins are recognized by the receptors. The transport complexes disassemble after translocation through the nuclear pore complex (NPC), and then the receptors return to the other side of the nuclear envelope. The receptors interact with proteins of the NPC (nucleoporins) and with the Ran GTPase, the major regulator of nucleocytoplasmic transport. Ran is an abundant protein that also shuttles between the nucleus and the cytoplasm. The Ranspecific GTPase activation protein (Rna1p, RanGAP1) is located in the cytoplasm, whereas the guanine nucleotide exchange factor (Prp20p, RCC1) is found in the nucleus. Accordingly, Ran should be bound to GTP in the nucleus and bound to GDP in the cytoplasm. This asymmetric distribution of Ran's nucleotide-bound state is thought to regulate cargo binding and release and, consequently, the transport direction of transport complexes (reviewed in references 13, 30, and 32). Import and export receptors both bind specifically to the GTPbound form of Ran (Ran-GTP) but respond in different ways. Import receptors bind to their substrates in the cytoplasm, where the concentration of Ran-GTP is low, and release them in the nucleus upon binding to Ran-GTP. The import receptor-Ran-GTP complexes then migrate...

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“…P450 52A3 and NADPHcytochrome P450 reductase (CPR) from C. maltosa were heterologously expressed in S. cerevisiae GRF18 and purified to electrophoretic homogeneity as described previously (17,20,21).…”

Section: Materials-[1-mentioning

confidence: 99%

Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

Scheller¹,

Zimmer²,

Becher³

et al. 1998

Journal of Biological Chemistry

155

119

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Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1,16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1,16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high V max values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min ؊1 , respectively), whereas the oxidation of hexadecanoic acid, 1,16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min ؊1 , respectively). 1-Hexadecanol was found to be the main branch point between mono-and diterminal oxidation. Taken together with data on the incorporation of 18 O 2 -derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono-and diterminal monooxygenation reactions from n-alkanes to ␣,-dioic acids with high regioselectivity.The capability of several yeast species to use n-alkanes and other aliphatic hydrocarbons as a sole source of carbon and energy is mediated by the existence of multiple microsomal cytochrome P450 forms. Corresponding P450 genes or cDNAs have been isolated from the alkane-assimilating yeasts Candida maltosa (1-6), C. tropicalis (7-9), and C. apicola (10). The presently available 21 sequences belong to the family CYP52, according to the nomenclature of the P450 superfamily (11).The metabolic functions of P450 in these yeasts have been established thus far in the terminal oxidation of long-chain n-alkanes to fatty alcohols as the first and rate-determining step of the n-alkane degradation pathway and in the -hydroxylation of fatty acids, initiating the diterminal degradation pathway (Refs. 12-14; for a review, see Ref. 15). Sequential gene disruption revealed that in C. maltosa, four of its eight CYP52 genes, namely CYP52A3, CYP52A4, CYP52A5, and CYP52A9, are directly involved in alkane assimilation (16). After heterologous expression in Saccharomyces cerevisiae, each of the corresponding P450 isoenzymes was found to exhibit an individual substrate specificity in terms of the preferred class (n-alkanes or fatty acids) and the chain length of the hydroxylated aliphatic compounds (17,18).In the present study, we addressed the question of whether P450 monooxygenases may be involved not only in the primary hydroxylation reactions mentioned above but also in the subsequent oxidation steps leading to the formation of fatty acids and long-chain ...

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Candida maltosa NADPH‐cytochrome P450 Reductase: Cloning of a Full‐length cDNA, Heterologous Expression in Saccharomyces cerevisiae and Function of the N‐terminal Region for Membrane Anchoring and Proliferation of the Endoplasmic Reticulum

Cited by 25 publications

References 0 publications

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol

Nup2p, a Yeast Nucleoporin, Functions in Bidirectional Transport of Importin α

Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

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