hem14 (SPAC1F5.07c; UniProt Q10062) encodes protoporphyrinogen oxidase (PPOX; EC 1.3.3.4), an FAD-containing enzyme of the PPOX subfamily. It carries out the six-electron, O2-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX, the seventh step of heme biosynthesis, and provides the substrate for ferrochelatase (hem15). Function and an inner mitochondrial membrane location are assigned by orthology to S. cerevisiae HEM14, whose activity is confined to the inner membrane; a genome-wide YFP screen in S. pombe instead recorded cytosolic and nuclear signal, and the location has not been tested directly.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PPOX node PTN000077911 (PTHR42923), with experimental seeds S. cerevisiae HEM14, mouse and rat PPOX and human PPOX. hem14 is in the PPOX subfamily (PTHR42923:SF3; TIGR00562); sound. Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt PANTHER; PTHR42923:SF3; PROTOPORPHYRINOGEN OXIDASE; 1. file:SCHPO/hem14/hem14-uniprot.txt FUNCTION: Catalyzes the 6-electron oxidation of protoporphyrinogen-IX to form protoporphyrin-IX. file:SCHPO/hem14/hem14-uniprot.txt Reaction=protoporphyrinogen IX + 3 O2 = protoporphyrin IX + 3 H2O2; |
| GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor | IEA GO_REF:0000120 | ACCEPT | Summary: EC/RHEA mapping; consistent. Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt Reaction=protoporphyrinogen IX + 3 O2 = protoporphyrin IX + 3 H2O2; |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | MARK AS OVER ANNOTATED | Summary: ORFeome YFP screen recorded nuclear signal (is_active_in nucleus) for hem14. No nuclear role is known for a membrane-bound PPOX. Reason: PPOX activity in S. cerevisiae is confined to the mitochondrial inner membrane, and the same S. pombe screen also reports extramitochondrial signal for ferrochelatase (hem15). An is_active_in claim from a C-terminally tagged, overexpressed high-throughput screen is not enough to place the activity outside mitochondria; an extramitochondrial pool is not ruled out, so the row is not removed. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins PMID:7798202 Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Mitochondrion, ECO:0000250 by similarity). Checked the source: it rests on S. cerevisiae Hem14, whose activity is confined to the mitochondrial inner membrane. The organelle claim is the orthology-based expectation; note that PAINT places an IRD NOT mitochondrial inner membrane at the S. pombe node (PTN000077950) and the S. pombe screen reports cytosolic signal, so the S. pombe location is untested. Accepted at the organelle level on orthology, with the open question recorded. Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000250}. PMID:7798202 Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). |
| GO:0005829 cytosol | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | MARK AS OVER ANNOTATED | Summary: ORFeome YFP screen recorded cytosolic signal (is_active_in cytosol). Eukaryotic PPOX is a mitochondrial inner-membrane enzyme, and a C-terminal YFP tag on an overexpressed protein can mask targeting. The observation is not disputed; the activity claim exceeds it. Resolved from UNDECIDED: the decision does not depend on the screen images, which are not cached. Reason: PPOX activity in S. cerevisiae is confined to the mitochondrial inner membrane, and the same S. pombe screen also reports extramitochondrial signal for ferrochelatase (hem15). An is_active_in claim from a C-terminally tagged, overexpressed high-throughput screen is not enough to place the activity outside mitochondria; an extramitochondrial pool is not ruled out, so the row is not removed. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins PMID:7798202 Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). |
| GO:0006779 porphyrin-containing compound biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to porphyrin-containing compound biosynthetic process, a parent of heme biosynthesis. Reason: Correct but general: heme biosynthetic process (GO:0006783), an is_a descendant already carried by hem14, is the process the enzyme acts in. Proposed replacements: heme biosynthetic process Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt FUNCTION: Catalyzes the 6-electron oxidation of protoporphyrinogen-IX to form protoporphyrin-IX. |
| GO:0006783 heme biosynthetic process | IC GO_REF:0000036 | ACCEPT | Summary: Seventh step of heme biosynthesis; curator inference from the activity. Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt FUNCTION: Catalyzes the 6-electron oxidation of protoporphyrinogen-IX to form protoporphyrin-IX. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: Generic oxidoreductase mapping; the specific activity is annotated. Reason: Correct but too general: protoporphyrinogen oxidase activity, oxygen as acceptor (GO:0004729), a descendant already carried by hem14, is the specific activity. Proposed replacements: protoporphyrinogen oxidase activity, oxygen as acceptor Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt Reaction=protoporphyrinogen IX + 3 O2 = protoporphyrin IX + 3 H2O2; |
| GO:0071949 FAD binding | ISM GO_REF:0000050 | KEEP AS NON CORE | Summary: FAD is the PPOX cofactor; correct but subsidiary. Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt Note=Binds 1 FAD per subunit. {ECO:0000250}; |
| GO:0099617 matrix side of mitochondrial inner membrane | IC GO_REF:0000024 | MODIFY | Summary: Curator inference placing PPOX on the matrix side of the inner membrane. Neither S. pombe nor the S. cerevisiae ortholog has a membrane-face assay in the cached literature (the yeast data show inner-membrane activity only), so the face is not established here; mitochondrial inner membrane is the level the ortholog evidence supports. Reason: Membrane face not established by cached evidence; generalise to the inner membrane supported by the ortholog. Proposed replacements: mitochondrial inner membrane Supporting Evidence: file:SCHPO/hem14/hem14-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000250}. PMID:7798202 Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). |
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Download this section (compressed HTML)Q: Is S. pombe hem14 exclusively mitochondrial, or does the cytosolic signal in the ORFeome screen reflect a real extramitochondrial pool, and on which face of the inner membrane is the enzyme?
Experiment: Tag hem14 at its endogenous locus (N- and C-terminal variants), image with a mitochondrial marker, and fractionate mitochondria (outer/inner membrane, protease protection) to test the location and membrane face; compare with untagged PPOX activity in fractions.
Hypothesis: S. pombe hem14 is an inner mitochondrial membrane enzyme, as its S. cerevisiae ortholog is
Type: cell biology / fractionation
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