id: A0A2U1PS28 gene_symbol: A0A2U1PS28 product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:35608 label: Artemisia annua description: 'Chloroplast-targeted elongation factor 4 (LepA, cpLEPA) of Artemisia annua, a ribosome-dependent translational GTPase of the LepA/GUF1 family. It belongs to the bacterial/chloroplast LepA subfamily rather than to the mitochondrial GUF1 subfamily, and is most similar to Arabidopsis cpLEPA (At5g08650). The 661-residue protein has an N-terminal extension with transit-peptide-like composition followed by the complete LepA architecture: a Tr-type G domain with P-loop and switch motifs, domains II, III and V shared with EF-G, and the unique LepA C-terminal domain. By homology with bacterial LepA, it binds translating 70S-type ribosomes in its GTP form, can back-translocate tRNAs by one codon and remodel the A-site tRNA, and hydrolyses GTP on the ribosome. In Arabidopsis the orthologous cpLEPA is a peripheral thylakoid-membrane protein with a smaller stromal pool, and its loss reduces chloroplast protein synthesis, photosynthetic activity and growth, especially under suboptimal conditions.' existing_annotations: - term: id: GO:0003924 label: GTPase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: Ribosome-dependent GTP hydrolysis is the catalytic activity of the LepA/EF4 family; the Tr-type G domain motifs, including the switch II DTPGH motif with catalytic His164, are intact. action: ACCEPT reason: Inherited from the LepA subfamily (PTHR43512:SF4, the same subfamily as E. coli LepA), in which ribosome-dependent multiple-turnover GTPase activity was measured directly (PMID:25712150). Core function. supported_by: - reference_id: PMID:25712150 supporting_text: successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G - reference_id: file:ARTAN/A0A2U1PS28/A0A2U1PS28-bioinformatics/RESULTS-rereview.md supporting_text: The G3/switch II motif DTPGH lies at 160-164 (catalytic His164). - term: id: GO:0005525 label: GTP binding evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: GTP binding by the Tr-type G domain is intrinsic to the LepA GTPase cycle; EF4 binds the ribosome in its GTP form. action: ACCEPT reason: Conserved G-domain nucleotide-binding motifs (UniProt binding sites 95-102, 160-164, 192-195) and structures of SF4 LepA bound to a GTP analogue on the ribosome (PMID:27092003). True and part of the core GTPase activity. supported_by: - reference_id: PMID:27092003 supporting_text: we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: 'Mitochondrial inner membrane localization is a misassignment: A0A2U1PS28 is the chloroplast LepA, not the mitochondrial GUF1. The essence of the row, a peripheral membrane-associated organellar translation factor, holds for the chloroplast thylakoid membrane.' action: MODIFY reason: This row comes from the UniProt SubCell term set by HAMAP rule MF_03137 (mitochondrial GUF1). A0A2U1PS28 is in PANTHER PTHR43512:SF4 (bacterial/chloroplast LepA) with Arabidopsis cpLEPA (82.9% identity), not in SF7 with yeast, human and Arabidopsis mitochondrial GUF1 (43-50% identity); the Arabidopsis ortholog is named by the chloroplastic rule MF_03138. PAINT placed mitochondrion only at the eukaryotic mitochondrial node, which the target does not descend from. Arabidopsis cpLEPA fractionates mainly with thylakoids and is released by carbonate/urea, i.e. a peripheral thylakoid membrane protein (PMID:23166764). Replace with chloroplast thylakoid membrane. supported_by: - &id001 reference_id: file:ARTAN/A0A2U1PS28/A0A2U1PS28-uniprot.txt record_section: ["DR:PANTHER"] supporting_text: DR PANTHER; PTHR43512:SF4; TRANSLATION FACTOR GUF1 HOMOLOG, CHLOROPLASTIC; 1. - &id002 reference_id: file:ARTAN/A0A2U1PS28/A0A2U1PS28-bioinformatics/RESULTS-rereview.md supporting_text: A0A2U1PS28 is assigned to SF4, the bacterial/chloroplast subfamily, not the mitochondrial SF7. - reference_id: PMID:23166764 supporting_text: most of the cpLEPA protein was detected in the thylakoid fractions - reference_id: PMID:23166764 supporting_text: These results indicate that cpLEPA is a membrane-associated protein. proposed_replacement_terms: - id: GO:0009535 label: chloroplast thylakoid membrane - term: id: GO:0005759 label: mitochondrial matrix evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: located_in review: summary: Mitochondrial matrix is the wrong organelle for this chloroplast LepA (HAMAP mitochondrial rule misapplied). The analogous soluble compartment is the chloroplast stroma, where a minor pool of the Arabidopsis ortholog is found. action: MODIFY reason: Propagated by UniRule from the mitochondrial GUF1 rule. PANTHER SF4 placement and sequence identity put A0A2U1PS28 with chloroplast cpLEPA. In Arabidopsis the stromal pool of cpLEPA is about a quarter of the thylakoid pool (PMID:23166764), so chloroplast stroma is the supported replacement. proposed_replacement_terms: - id: GO:0009570 label: chloroplast stroma supported_by: - *id001 - *id002 - reference_id: PMID:23166764 supporting_text: the ratio of cpLEPA in the stroma to cpLEPA in the thylakoid membrane was approximately 0.25 - term: id: GO:0043022 label: ribosome binding evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: Ribosome binding is the defining property of LepA/EF4, which binds pre- and post-translocation ribosomes in its GTP form; here, chloroplast ribosomes. action: ACCEPT reason: PAINT placed ribosome binding at PTN002460656 with E. coli LepA (SF4) among the seeds, a node the target inherits from; structures of SF4 LepA on the ribosome show extensive rRNA and tRNA contacts (PMID:27092003, PMID:27137929). Core function. supported_by: - reference_id: PMID:27137929 supporting_text: we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution - reference_id: PMID:27092003 supporting_text: The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA - term: id: GO:0045727 label: positive regulation of translation evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in review: summary: LepA family members promote translation efficiency, mainly under suboptimal conditions; Arabidopsis cpLEPA promotes the efficiency of chloroplast protein synthesis. action: KEEP_AS_NON_CORE reason: The term comes from the PAINT node PTN002460656 (seeded by yeast Guf1), which the target inherits from, and it is consistent with the conditional enhancement of chloroplast protein synthesis by the Arabidopsis ortholog (PMID:23166764). It is a regulatory reading of what is better described as direct participation in plastid translation, so it is kept as non-core. supported_by: - reference_id: PMID:23166764 supporting_text: promotes the efficiency of chloroplast protein synthesis - term: id: GO:0032544 label: plastid translation evidence_type: ISS original_reference_id: PMID:23166764 qualifier: involved_in review: summary: The protein is a chloroplast translation factor that acts on the ribosome during protein synthesis in the plastid. action: NEW reason: Replaces the previous NEW 'translation' row, which was supported by the yeast mitochondrial Guf1 paper (SF7, the other subfamily). LepA performs a step of translation itself (GTP-dependent tRNA back-translocation/remodelling on the ribosome), so this is participation, not mere requirement. The SF4 ortholog cpLEPA (82.9% identity) is required for efficient chloroplast protein synthesis and polysome loading of plastid mRNAs (PMID:23166764). GO:0032544 is not in GOA for this entry; the UniProt KW-derived 'translation' line is its unspecific ancestor. supported_by: - reference_id: PMID:23166764 supporting_text: promotes the efficiency of chloroplast protein synthesis - *id002 references: - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000104 title: Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:25712150 title: Identification of two structural elements important for ribosome-dependent GTPase activity of elongation factor 4 (EF4/LepA). findings: - statement: EF4 has ribosome-dependent multiple turnover GTPase activity similar to EF-G, and the conserved histidine His81 is essential for this activity. supporting_text: successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Bacterial EF4/LepA biochemistry or structure; LepA is in the same PANTHER subfamily (SF4) as the target. - id: PMID:27137929 title: Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome. findings: - statement: Cryo-EM structure of EF4-GTP-ribosome complex at 3.8 A resolution reveals the GTPase activation mechanism. supporting_text: we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Bacterial EF4/LepA biochemistry or structure; LepA is in the same PANTHER subfamily (SF4) as the target. - id: PMID:18442968 title: The membrane-bound GTPase Guf1 promotes mitochondrial protein synthesis under suboptimal conditions. findings: - statement: Yeast Guf1 (mitochondrial GUF1 subfamily, PTHR43512:SF7) binds mitochondrial ribosomes in a GTP-dependent manner and promotes mitochondrial protein synthesis. supporting_text: It binds to mitochondrial ribosomes in a GTP-dependent manner reference_review: relevance: LOW correctness: VERIFIED review_notes: Correct paper, but it concerns the mitochondrial GUF1 subfamily (SF7), not the chloroplast LepA subfamily (SF4) of A0A2U1PS28; no longer used as support, kept as background on the family. - id: PMID:27092003 title: Elongation factor 4 remodels the A-site tRNA on the ribosome. findings: - statement: Crystal structure at 2.6 A shows EF-4 bound to the ribosome with tRNAs, revealing the C-terminal domain contacts with the A-site tRNA acceptor stem. supporting_text: we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Bacterial EF4/LepA biochemistry or structure; LepA is in the same PANTHER subfamily (SF4) as the target. - id: PMID:23166764 title: CpLEPA is critical for chloroplast protein synthesis under suboptimal conditions in Arabidopsis thaliana. findings: - statement: Arabidopsis cpLEPA (At5g08650), the SF4 ortholog, is required for efficient chloroplast protein synthesis. supporting_text: promotes the efficiency of chloroplast protein synthesis - statement: cpLEPA is a peripheral thylakoid membrane-associated protein with a minor stromal pool. supporting_text: most of the cpLEPA protein was detected in the thylakoid fractions reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached; primary characterisation of the closest experimentally studied ortholog. - id: PMID:18431481 title: Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Chloroplast proteome study behind the HDA chloroplast annotation of Arabidopsis Q9FNM5 (QuickGO); background only, not quoted. - id: file:ARTAN/A0A2U1PS28/A0A2U1PS28-bioinformatics/RESULTS-rereview.md title: 'A0A2U1PS28 re-review: placement relative to characterised LepA/GUF1 proteins' findings: [] - id: file:ARTAN/A0A2U1PS28/A0A2U1PS28-uniprot.txt title: UniProt entry A0A2U1PS28 findings: [] core_functions: - description: 'Ribosome-dependent translational GTPase of the chloroplast LepA subfamily: in its GTP form it binds translating chloroplast ribosomes, can back-translocate or remodel tRNAs, and hydrolyses GTP, promoting efficient plastid protein synthesis.' molecular_function: id: GO:0003924 label: GTPase activity directly_involved_in: - id: GO:0032544 label: plastid translation locations: - id: GO:0009535 label: chloroplast thylakoid membrane - id: GO:0009570 label: chloroplast stroma supported_by: - reference_id: PMID:25712150 supporting_text: successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G - reference_id: PMID:23166764 supporting_text: promotes the efficiency of chloroplast protein synthesis - *id001 - *id002 - description: Binds pre- and post-translocation chloroplast 70S-type ribosomes, with its unique C-terminal domain contacting the acceptor stem of the A-site tRNA. molecular_function: id: GO:0043022 label: ribosome binding directly_involved_in: - id: GO:0032544 label: plastid translation locations: - id: GO:0009535 label: chloroplast thylakoid membrane supported_by: - reference_id: PMID:27092003 supporting_text: The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA - reference_id: PMID:27137929 supporting_text: we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution - *id002 proposed_new_terms: [] suggested_questions: - question: Should UniProt reassign A0A2U1PS28 from HAMAP MF_03137 (mitochondrial GUF1) to MF_03138 (chloroplastic GUF1), given its PANTHER SF4 placement and 83% identity to Arabidopsis cpLEPA? experts: [] - question: Is there a GO molecular function term suitable for ribosome back-translocation by LepA/EF4, more specific than GTPase activity? experts: [] - question: Is chloroplast LepA bound to thylakoid membranes through thylakoid-associated ribosomes or through a membrane partner, as proposed for mitochondrial Guf1 at the inner membrane? experts: [] suggested_experiments: - description: Express A0A2U1PS28-GFP in A. annua or Nicotiana benthamiana leaves and image with chloroplast autofluorescence and a mitochondrial marker; fractionate chloroplasts into stroma and thylakoids. hypothesis: A0A2U1PS28 is chloroplast-targeted and mainly thylakoid-associated, like Arabidopsis cpLEPA. - description: Complement the Arabidopsis cplepa-1 mutant with A0A2U1PS28 and measure chloroplast protein synthesis by in vivo labelling and polysome loading under high light. hypothesis: A0A2U1PS28 is functionally equivalent to Arabidopsis cpLEPA.