NCP1 (YHR042W) encodes NADPH--cytochrome P450 reductase (CPR, EC 1.6.2.4), a diflavin reductase that binds FAD and FMN and transfers electrons from NADPH to microsomal cytochrome P450 enzymes and other hemoproteins. It is anchored by an N-terminal transmembrane segment in the endoplasmic reticulum membrane with its catalytic domain facing the cytoplasm, and is also detected in mitochondrial outer membrane and plasma membrane fractions. NCP1 supplies electrons to the ergosterol-pathway oxygenases squalene epoxidase (Erg1), lanosterol 14alpha-demethylase (Erg11/CYP51) and sterol C-22 desaturase (Erg5/CYP61). Deletion is viable but lowers ergosterol and makes cells highly sensitive to azole antifungals; cytochrome b5 (CYB5) provides an alternative electron supply. NCP1 also accounts for the NADPH-dependent ferrireductase activity of isolated plasma membranes and can reduce Dph3 in vitro. Purified yeast NCP1 reductase can also supply electrons to the heme oxygenase Hmx1 in vitro, although ascorbate is a more effective reductant. NCP1 expression is coordinately regulated with that of its main P450 partner ERG11.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003958 NADPH-hemoprotein reductase activity | EXP PMID:11485306 Activities and kinetic mechanisms of native and soluble NADP... | ACCEPT | Summary: Purified recombinant Ncp1 (native and soluble forms) reduces cytochrome c and reconstitutes CYP61 sterol 22-desaturation with NADPH (PMID:11485306), directly demonstrating NADPH-hemoprotein reductase activity. Reason: Core MF, direct biochemical evidence. Supporting Evidence: PMID:11485306 Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Delta(22)-desaturation |
| GO:0003958 NADPH-hemoprotein reductase activity | EXP PMID:9368374 Cytochrome P-450 reductase is responsible for the ferrireduc... | ACCEPT | Summary: Ncp1 accounts for the NADPH-dependent reductase (ferrireductase) activity of isolated plasma membranes (PMID:9368374). Consistent with its NADPH-hemoprotein/one-electron reductase activity. Reason: Experimental support for the reductase activity, from the curator's EXP assignment. Supporting Evidence: PMID:9368374 Cytochrome P-450 reductase (encoded by the NCP1 gene) was found to catalyse all the NADPH-dependent ferrireductase activities associated with isolated plasma membranes |
| GO:0003958 NADPH-hemoprotein reductase activity | EXP PMID:9468503 The N-terminal membrane domain of yeast NADPH-cytochrome P45... | ACCEPT | Summary: Purified native and N-terminally truncated Ncp1 reconstitute CYP61 and CYP51 activities and ergosterol biosynthesis in a cell-free system (PMID:9468503). Reason: Core MF, direct reconstitution evidence. Supporting Evidence: PMID:9468503 Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system |
| GO:0003958 NADPH-hemoprotein reductase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from the CPR clade (PTN000453765; human POR, rat Por, plant ATR1/2, S. pombe ccr1 and yeast NCP1 itself). Ncp1 is an orthodox member of this clade. Reason: Sound phylogenetic inference matching experimental data for Ncp1. Supporting Evidence: PMID:11485306 Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Delta(22)-desaturation |
| GO:0003958 NADPH-hemoprotein reductase activity | IDA PMID:9087488 Characterization of Saccharomyces cerevisiae CYP51 and a CYP... | ACCEPT | Summary: A CYP51-Ncp1 fusion protein expressed in E. coli catalysed NADPH-dependent 14alpha-demethylation, whereas CYP51 alone did not, showing that the reductase supplies electrons to CYP51 (PMID:9087488). Reason: Direct functional evidence of P450 reduction by Ncp1. Supporting Evidence: PMID:9087488 FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH |
| GO:0003958 NADPH-hemoprotein reductase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (UniRule/ARBA, EC:1.6.2.4, RHEA:24040) for NADPH-hemoprotein reductase. Correct for Ncp1. Reason: Correct EC-based mapping, consistent with experimental data. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Reaction=2 oxidized [cytochrome P450] + NADPH = 2 reduced [cytochrome |
| GO:0003959 NADPH dehydrogenase activity | IDA PMID:27694803 Cbr1 is a Dph3 reductase required for the tRNA wobble uridin... | KEEP AS NON CORE | Summary: Recombinant Ncp1 reduced Dph3 with NADPH in vitro and supported the first step of diphthamide formation, more slowly than Cbr1 (PMID:27694803). The activity is real in vitro but the in vivo Dph3 reductase is Cbr1 and the relevance for Ncp1 is not shown. Reason: In vitro NADPH-dependent reduction of a non-P450 acceptor; consistent with a promiscuous diflavin reductase but not a demonstrated physiological role. Supporting Evidence: PMID:27694803 both Mcr1 and Ncp1 reduced Dph3 at a slower rate compared to that of Cbr1 under similar reaction conditions |
| GO:0005737 cytoplasm | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule-based cytoplasm. Ncp1 is a membrane protein whose catalytic domain faces the cytoplasm; the term is technically true (ER is part of the cytoplasm) but uninformative. Reason: Correct though general; ER membrane is the informative location. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane |
| GO:0005739 mitochondrion | HDA PMID:14576278 The proteome of Saccharomyces cerevisiae mitochondria. | KEEP AS NON CORE | Summary: Detected in the proteome of purified mitochondria (PMID:14576278). Ncp1 is mainly ER-resident; detection on the mitochondrial outer membrane is real but secondary. Reason: Minor/secondary location from proteomics. Supporting Evidence: PMID:14576278 The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS |
| GO:0005739 mitochondrion | HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen... | KEEP AS NON CORE | Summary: High-throughput mitochondrial proteomics detection (PMID:16823961). Secondary location of an ER-anchored reductase. Reason: Secondary location from proteomics. Supporting Evidence: PMID:16823961 Toward the complete yeast mitochondrial proteome |
| GO:0005739 mitochondrion | HDA PMID:24769239 Quantitative variations of the mitochondrial proteome and ph... | KEEP AS NON CORE | Summary: Quantitative mitochondrial proteomics detection (PMID:24769239). Secondary location of an ER-anchored reductase. Reason: Secondary location from proteomics. Supporting Evidence: PMID:24769239 Quantitative variations of the mitochondrial proteome and phosphoproteome |
| GO:0005739 mitochondrion | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule mitochondrion, reflecting the mitochondrial outer membrane detection recorded by UniProt. Secondary location. Reason: Secondary location; ER membrane is primary. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Mitochondrion outer membrane |
| GO:0005741 mitochondrial outer membrane | EXP PMID:14576278 The proteome of Saccharomyces cerevisiae mitochondria. | KEEP AS NON CORE | Summary: Mitochondrial outer membrane assignment from mitochondrial proteomics (PMID:14576278), recorded by UniProt. Real but secondary to ER. Reason: Secondary location. Supporting Evidence: PMID:14576278 The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS |
| GO:0005741 mitochondrial outer membrane | HDA PMID:16407407 Proteomic analysis of the yeast mitochondrial outer membrane... | KEEP AS NON CORE | Summary: Found in the proteome of highly purified mitochondrial outer membranes (PMID:16407407). Secondary location. Reason: Secondary location from proteomics. Supporting Evidence: PMID:16407407 to determine the proteome of highly purified mitochondrial outer membranes |
| GO:0005741 mitochondrial outer membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular-location mapping (SL-0172) of the mitochondrial outer membrane detection. Reason: Secondary location. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Mitochondrion outer membrane |
| GO:0005789 endoplasmic reticulum membrane | EXP PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: Genome-wide GFP localisation (PMID:14562095) places Ncp1 in the ER, its main site of action with ER P450s. Reason: Core location. Supporting Evidence: PMID:14562095 Global analysis of protein localization in budding yeast |
| GO:0005789 endoplasmic reticulum membrane | EXP PMID:9468503 The N-terminal membrane domain of yeast NADPH-cytochrome P45... | ACCEPT | Summary: Native Ncp1 is microsomal/ER membrane-anchored; removing the N-terminal 33 residues made it cytosolic yet still functional (PMID:9468503). Reason: Core location. Supporting Evidence: PMID:9468503 a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated ER membrane annotation (SL-0097, UniRule); correct. Reason: Core location. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane |
| GO:0005829 cytosol | IBA GO_REF:0000033 | REMOVE | Summary: IBA "is_active_in cytosol" from a broad diflavin-reductase node (PTN000453956) seeded by cytosolic members such as nitric oxide synthase (P35228), methionine synthase reductase (Q9UBK8), NDOR1 (Q9UHB4) and yeast Tah18 (SGD:S000006252). Native Ncp1 is a membrane-anchored ER protein; it is only soluble when its N-terminal anchor is artificially removed (PMID:9468503). Reason: The cytosolic location belongs to the soluble diflavin reductase subfamilies, not to membrane-anchored CPR; over-propagation to Ncp1. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN000453956 · PTN000453956 SUPPORTS SOURCE BUT NOT TARGET Cytosolic location is supported for soluble NOS/MTRR/NDOR1/Tah18 members but CPR carries an N-terminal ER membrane anchor. Supporting Evidence: PMID:9468503 a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol file:yeast/NCP1/NCP1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane |
| GO:0005886 plasma membrane | EXP PMID:9368374 Cytochrome P-450 reductase is responsible for the ferrireduc... | KEEP AS NON CORE | Summary: Ncp1 accounts for the NADPH-dependent ferrireductase activity of isolated plasma membrane preparations (PMID:9368374). Plasma membrane is a minor, secondary location. Reason: Secondary location linked to a non-core ferrireductase role. Supporting Evidence: PMID:9368374 Cytochrome P-450 reductase (encoded by the NCP1 gene) was found to catalyse all the NADPH-dependent ferrireductase activities associated with isolated plasma membranes |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Automated cell membrane mapping (SL-0039) of the plasma membrane detection. Secondary location. Reason: Secondary location. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Cell membrane |
| GO:0006696 ergosterol biosynthetic process | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule ergosterol biosynthetic process for fungal CPR; consistent with experimental data. Reason: Core BP. Supporting Evidence: PMID:9468503 function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway |
| GO:0006696 ergosterol biosynthetic process | IMP PMID:9468503 The N-terminal membrane domain of yeast NADPH-cytochrome P45... | ACCEPT | Summary: ncp1 disruption lowers ergosterol to ~25% of wild type; Ncp1 supplies electrons to Erg1, Erg11 and Erg5 (PMID:9468503). Ncp1 does work in the process by reducing the pathway oxygenases. Reason: Core BP. Supporting Evidence: PMID:9468503 resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent |
| GO:0009055 electron transfer activity | IDA PMID:11485306 Activities and kinetic mechanisms of native and soluble NADP... | MODIFY | Summary: Electron transfer from NADPH to cytochrome c / CYP61 (PMID:11485306). Ncp1 is an enzyme (oxidoreductase), not a simple electron carrier; the catalysed reaction is captured precisely by NADPH-hemoprotein reductase activity. Reason: The more specific enzymatic term describes the measured activity. Proposed replacements: NADPH-hemoprotein reductase activity Supporting Evidence: PMID:11485306 Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with NADPH donating electrons directly to CPR |
| GO:0010181 FMN binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA FMN binding for the diflavin reductase family; Ncp1 binds one FMN per monomer. Reason: Correct cofactor binding. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Note=Binds 1 FMN per monomer. |
| GO:0010181 FMN binding | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro flavodoxin-domain mapping to FMN binding; correct for Ncp1. Reason: Correct cofactor binding. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Note=Binds 1 FMN per monomer. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro-based generic oxidoreductase activity; true but the specific activity (EC 1.6.2.4) is known. Reason: Replace with the specific NADPH-hemoprotein reductase activity. Proposed replacements: NADPH-hemoprotein reductase activity Supporting Evidence: PMID:11485306 Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Delta(22)-desaturation |
| GO:0050660 flavin adenine dinucleotide binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA FAD binding for diflavin reductases; Ncp1 binds one FAD per monomer. Reason: Correct cofactor binding. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Note=Binds 1 FAD per monomer. |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule FAD binding; correct. Reason: Correct cofactor binding. Supporting Evidence: file:yeast/NCP1/NCP1-uniprot.txt Note=Binds 1 FAD per monomer. |
| GO:0050661 NADP binding | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule NADP binding; NADPH is the electron donor of CPR. Reason: Correct substrate/cofactor binding. Supporting Evidence: PMID:11485306 NADPH donating electrons directly to CPR to produce a reduced intermediary form of the enzyme |
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Download this section (compressed HTML)Q: How is electron supply to Erg11 partitioned between Ncp1 and the Cyb5/Cbr1 route in wild-type cells, and does the mitochondrial outer membrane pool of Ncp1 support any P450 or other reductive activity there?
Experiment: Quantify sterol intermediates and azole sensitivity in ncp1, cyb5, cbr1 and combined mutants, and test an ER-restricted versus MOM-targeted Ncp1 for complementation, to separate the contributions of each electron route and compartment.
NADP-cytochrome P450 reductase; involved in ergosterol biosynthesis; associated and coordinately regulated with Erg11p
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