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.
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.
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.
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
Summary: GTP binding by the Tr-type G domain is intrinsic to the LepA GTPase cycle; EF4 binds the ribosome in its GTP form.
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.
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
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.
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.
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.
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.
Summary: Ribosome binding is the defining property of LepA/EF4, which binds pre- and post-translocation ribosomes in its GTP form; here, chloroplast ribosomes.
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.
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
Summary: LepA family members promote translation efficiency, mainly under suboptimal conditions; Arabidopsis cpLEPA promotes the efficiency of chloroplast protein synthesis.
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.
ISS PMID:23166764 CpLEPA is critical for chloroplast protein synthesis under s...
NEW
Summary: The protein is a chloroplast translation factor that acts on the ribosome during protein synthesis in the plastid.
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.
A0A2U1PS28 is assigned to SF4, the bacterial/chloroplast subfamily, not the mitochondrial SF7.
Core Functions
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.
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
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
Q: 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?
Q: Is there a GO molecular function term suitable for ribosome back-translocation by LepA/EF4, more specific than GTPase activity?
Q: 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?
Suggested Experiments
Experiment: 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.
Experiment: 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.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Chloroplast LepA/EF4 translational GTPase of Artemisia annua (PANTHER PTHR43512:SF4, ortholog of Arabidopsis cpLEPA). The single ProtNLM2 prediction, chloroplast, is a correct novel localization supported by subfamily transfer from the experimentally localized Arabidopsis ortholog.
Prediction method: ProtNLM2 · Version: UniProt 2024_06 pilot
Review rationale: The 661-residue target has the complete EF4/LepA architecture and is placed in the chloroplastic LepA subfamily PTHR43512:SF4, sharing 82.9% identity with Arabidopsis cpLEPA (At5g08650) and clearly less with the mitochondrial GUF1 subfamily SF7. Arabidopsis cpLEPA was shown by fractionation of intact chloroplasts to reside mainly on thylakoids with a minor stromal pool, and its loss impairs chloroplast protein synthesis (PMID:23166764), which grounds transfer of chloroplast localization within the plant SF4 branch. The re-reviewed gene review agrees, replacing the HAMAP-derived mitochondrial inner membrane and matrix rows with chloroplast thylakoid membrane and chloroplast stroma; those mitochondrial rows are the only cellular-component annotations in GOA, so chloroplast is novel for this gene. The term is correct though less specific than the suborganellar locations the evidence supports, and it rests on subfamily transfer rather than a targeting assay on the Artemisia protein.
Supporting Evidence:
file:ARTAN/A0A2U1PS28/A0A2U1PS28-uniprot.txt: "ID A0A2U1PS28_ARTAN Unreviewed; 661 AA. ... DR InterPro; IPR006297; EF-4. ... DR PANTHER; PTHR43512:SF4; TRANSLATION FACTOR GUF1 HOMOLOG, CHLOROPLASTIC; 1. ... FT DOMAIN 86..245 ... FT /note="Tr-type G""
PMID:23166764: "promotes the efficiency of chloroplast protein synthesis"
PMID:23166764: "intact chloroplasts were isolated and fractionated, and the proteins were subjected to immunoblot analysis with a specific cpLEPA antibody"
PMID:23166764: "most of the cpLEPA protein was detected in the thylakoid fractions"