ArnF (formerly PmrM/YfbJ) is a subunit of the undecaprenyl phosphate-alpha-L-Ara4N flippase, forming a heterodimer with ArnE that translocates 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol (alpha-L-Ara4N-phosphoundecaprenol) from the cytoplasmic to the periplasmic side of the inner membrane [PMID:17928292]. This flipping step is essential for L-Ara4N modification of lipid A by ArnT (whose active site faces the periplasm), which confers resistance to polymyxin and cationic antimicrobial peptides. ArnF is a 128 AA inner membrane protein with 4 transmembrane helices, distantly related to EmrE and other members of the DMT/SMR superfamily. It is part of the arnBCADTEF operon, regulated by BasR in response to iron and other environmental stimuli [PMID:15569938].
Definition: Enables the directional transport of a lipid from one leaflet of a membrane to the other leaflet, without coupling to ATP hydrolysis. Unlike scramblases, the transport is directional and substrate-specific.
Justification: The current GO hierarchy under GO:0140303 (intramembrane lipid transporter activity) has two children -- ATPase-coupled intramembrane lipid transporter activity (GO:0140326, parent of flippase/floppase) and phospholipid scramblase activity (GO:0017128, ATP-independent but non-selective/bidirectional). ArnE/ArnF and possibly other bacterial undecaprenyl-linked sugar flippases are directional and substrate-specific but not ATP-dependent, falling in a gap between these two branches. GO:0015161 (lipid III floppase activity) is the closest analog but is classified as ATP-dependent under floppase activity.
Parent term: intramembrane lipid transporter activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0022857 transmembrane transporter activity | IBA GO_REF:0000033 | MODIFY | Summary: ArnF is a subunit of the undecaprenyl phosphate-alpha-L-Ara4N flippase, which translocates a lipid-linked sugar across the inner membrane [PMID:17928292]. This is technically an intramembrane lipid transporter (flipping a lipid-linked substrate from one leaflet to the other), rather than a classical transmembrane transporter that moves solutes across the membrane. The IBA annotation to the parent term 'transmembrane transporter activity' captures the general transporter function, which is phylogenetically sound given the SMR/DMT superfamily membership. Reason: While ArnF is part of the transporter superfamily (DMT/SMR) and the IBA annotation is phylogenetically reasonable, 'transmembrane transporter activity' (GO:0022857) is too generic and does not capture the specific intramembrane lipid flipping function. The more accurate term is GO:0140303 'intramembrane lipid transporter activity', which describes transport of a lipid from one region of a membrane to a different region on the same membrane -- precisely the flippase function demonstrated for ArnF [PMID:17928292]. Proposed replacements: intramembrane lipid transporter activity Supporting Evidence: PMID:17928292 PmrL and PmrM could specifically function to flip undecaprenyl phosphate-alpha-L-Ara4N from the cytosolic to the periplasmic side of the inner membrane, possibly functioning as a heterodimer |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: ArnF is an inner membrane protein in E. coli. In GO, the E. coli inner membrane (cytoplasmic membrane) is annotated as 'plasma membrane' (GO:0005886), which is correct usage for bacteria. This is supported by experimental evidence from topology analysis [PMID:15919996] and is consistent with its function as a flippase in the inner membrane [PMID:17928292]. Reason: The IBA annotation to plasma membrane is correct. ArnF is an integral inner membrane protein with 4 transmembrane helices, confirmed by global topology analysis. In bacterial GO annotation, the inner/cytoplasmic membrane is properly annotated as plasma membrane (GO:0005886). Supporting Evidence: PMID:15919996 Global topology analysis of the Escherichia coli inner membrane proteome PMID:17928292 PmrL and PmrM are small, hydrophobic, inner membrane proteins with many of the characteristics of small multidrug resistance transporters. Both PmrL and PmrM are predicted to have four membrane-spanning helices |
| GO:0055085 transmembrane transport | IBA GO_REF:0000033 | ACCEPT | Summary: ArnF participates in the translocation of undecaprenyl phosphate-alpha-L-Ara4N across the inner membrane [PMID:17928292]. This is an intramembrane translocation (flipping from one leaflet to the other) rather than a transmembrane transport event in the classical sense of moving a solute from one aqueous compartment to another. The IBA annotation captures the general transport process from the SMR family phylogenetic context. Reason: While the precise mechanism is intramembrane lipid flipping rather than classical transmembrane transport of a solute, GO:0055085 'transmembrane transport' is a reasonable process term for this function since the substrate does cross the membrane bilayer. The IBA inference from the SMR/DMT family is phylogenetically sound. The existing experimental IMP annotations to more specific terms (GO:1901264 carbohydrate derivative transport) complement this broader annotation. Supporting Evidence: PMID:17928292 The data imply that both PmrL and PmrM are involved in the translocation of undecaprenyl phosphate-alpha-LAra4N across the inner membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Automated annotation to plasma membrane. ArnF is confirmed as an inner membrane protein (equivalent to plasma membrane in bacteria) by both experimental topology studies [PMID:15919996] and by its established function as a flippase in the inner membrane [PMID:17928292]. Reason: This IEA annotation is correct and is independently supported by IDA evidence (PMID:15919996) and IBA evidence (GO_REF:0000033). Duplicating the more specific experimental annotation is acceptable for IEA. Supporting Evidence: PMID:15919996 Global topology analysis of the Escherichia coli inner membrane proteome |
| GO:0006629 lipid metabolic process | IEA GO_REF:0000043 | ACCEPT | Summary: Automated annotation from UniProt keyword mapping. ArnF's primary function is as a flippase for lipid-linked sugar substrates, participating in the L-Ara4N lipid A modification pathway [PMID:17928292]. While ArnF is involved in the overall pathway of lipid A modification, calling its specific function 'lipid metabolic process' is very broad. Reason: While this is a very broad term, it is not incorrect. ArnF participates in a lipid metabolic pathway (lipid A modification) by translocating a lipid-linked substrate. The UniProt keywords include 'Lipid biosynthesis' and 'Lipid metabolism' which map to this term. More specific BP annotations (GO:0046493 lipid A metabolic process, GO:0009245 lipid A biosynthetic process) are also present and provide the necessary specificity. This IEA serves as a broader parent annotation. Supporting Evidence: PMID:17928292 We propose that PmrL and PmrM be renamed ArnE and ArnF, respectively (Fig. 2B), given their function in generating L-Ara4N modified lipid A species |
| GO:0009103 lipopolysaccharide biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Automated annotation to LPS biosynthesis. ArnF is part of the arn operon which is involved in modifying lipid A (the lipid component of LPS) with L-Ara4N [PMID:17928292]. UniProt explicitly lists this under PATHWAY as 'Bacterial outer membrane biogenesis; lipopolysaccharide biosynthesis'. Reason: ArnF functions in the L-Ara4N modification of lipid A, which is a component of lipopolysaccharide. The LPS biosynthetic process term appropriately captures the pathway context. This is consistent with the UniProt pathway annotation and the established role of the arn operon in LPS modification. Supporting Evidence: PMID:17928292 Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium |
| GO:0009245 lipid A biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Automated annotation to lipid A biosynthesis. ArnF participates in the L-Ara4N modification of lipid A, which is a post-biosynthetic modification rather than de novo lipid A biosynthesis [PMID:17928292]. The more precise term would be GO:0046493 'lipid A metabolic process' (which is already annotated via IMP), since ArnF is involved in modifying existing lipid A rather than synthesizing it de novo. Reason: While strictly speaking ArnF participates in lipid A modification rather than de novo lipid A biosynthesis, the L-Ara4N modification pathway is conventionally grouped under lipid A biosynthesis in the broader sense of generating the final modified lipid A species. UniProt keywords include 'Lipid A biosynthesis', and this annotation is broadly consistent. The IMP annotation to GO:0046493 (lipid A metabolic process) provides a more accurate complementary annotation. Supporting Evidence: PMID:17928292 In the pmrL or pmrM deletion mutants, over 95% of each of the L-Ara4N-modified lipid A species was missing |
| GO:0016020 membrane | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based annotation to the generic 'membrane' component. ArnF is an integral membrane protein with 4 transmembrane helices located in the inner (plasma) membrane. This is correct but much less specific than the plasma membrane annotations already present. Reason: This is a correct but very broad cellular component annotation. ArnF is unquestionably a membrane protein (4 TM helices, integral inner membrane protein). While GO:0005886 (plasma membrane) is more informative and is already annotated via IBA, IDA, and IEA, keeping this broader IEA annotation is acceptable as it provides the InterPro-derived evidence. Supporting Evidence: PMID:17928292 A BLASTp analysis of PmrL and PmrM shows that both are small integral membrane proteins with four putative trans-membrane helices |
| GO:0022857 transmembrane transporter activity | IEA GO_REF:0000120 | MODIFY | Summary: Automated annotation to transmembrane transporter activity. ArnF is a subunit of the undecaprenyl phosphate-alpha-L-Ara4N flippase [PMID:17928292], which functions as an intramembrane lipid transporter rather than a classical transmembrane transporter. This IEA parallels the IBA annotation to the same term. Reason: This IEA annotation to GO:0022857 is the same term as the IBA annotation. As with the IBA, 'transmembrane transporter activity' is too generic for ArnF's specific intramembrane lipid flipping function. The more accurate term is GO:0140303 'intramembrane lipid transporter activity', which precisely describes the flippase function demonstrated for ArnF [PMID:17928292]. Proposed replacements: intramembrane lipid transporter activity Supporting Evidence: PMID:17928292 PmrL and PmrM could specifically function to flip undecaprenyl phosphate-alpha-L-Ara4N from the cytosolic to the periplasmic side of the inner membrane, possibly functioning as a heterodimer |
| GO:0055085 transmembrane transport | IEA GO_REF:0000104 | ACCEPT | Summary: Automated annotation to transmembrane transport, transferred from manual annotations on related proteins via shared sequence features. This parallels the IBA annotation to the same BP term and is broadly consistent with ArnF's role in translocating undecaprenyl phosphate-alpha-L-Ara4N across the inner membrane [PMID:17928292]. Reason: This IEA annotation is consistent with the IBA annotation to the same term and is supported by the experimental evidence for ArnF's flippase function. As an automated annotation it provides an acceptable broader process term. Supporting Evidence: PMID:17928292 The data imply that both PmrL and PmrM are involved in the translocation of undecaprenyl phosphate-alpha-LAra4N across the inner membrane |
| GO:1901505 carbohydrate derivative transmembrane transporter activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based annotation to carbohydrate derivative transmembrane transporter activity. The substrate of ArnF is undecaprenyl phosphate-alpha-L-Ara4N, which contains L-Ara4N (4-amino-4-deoxy-L-arabinose), a carbohydrate derivative. This term is more specific than generic transmembrane transporter activity and captures the carbohydrate-linked nature of the substrate [PMID:17928292]. Reason: This annotation is accurate -- the substrate translocated by the ArnE/ArnF flippase is undecaprenyl phosphate-alpha-L-Ara4N, which is a carbohydrate derivative (containing L-Ara4N, a modified arabinose). This is more informative than the generic 'transmembrane transporter activity' and is consistent with the IMP annotation to the same term from PMID:17928292. Supporting Evidence: PMID:17928292 PmrL and PmrM could specifically function to flip undecaprenyl phosphate-alpha-L-Ara4N from the cytosolic to the periplasmic side of the inner membrane |
| GO:0046493 lipid A metabolic process | IMP PMID:17928292 An undecaprenyl phosphate-aminoarabinose flippase required f... | ACCEPT | Summary: In-frame deletion of arnF (pmrM) in a pmrAc background results in loss of over 95% of L-Ara4N-modified lipid A species, demonstrating that ArnF is required for lipid A modification [PMID:17928292]. The mutant phenotype (loss of lipid A modification, polymyxin sensitivity) directly implicates ArnF in the lipid A metabolic process. Reason: This IMP annotation is well-supported by the deletion phenotype data in Yan et al. 2007. The arnF deletion mutant lacks L-Ara4N modified lipid A, demonstrating a clear role in lipid A metabolism. This is a core process annotation for ArnF. Supporting Evidence: PMID:17928292 In the pmrL or pmrM deletion mutants, over 95% of each of the L-Ara4N-modified lipid A species was missing PMID:17928292 the pmrL and the pmrM deletion mutants (as well as the double deletion mutant) were sensitive to 15 ΞΌg/ml polymyxin |
| GO:0010041 response to iron(III) ion | IGI PMID:12139617 Fe(III)-mediated cellular toxicity. | MARK AS OVER ANNOTATED | Summary: This annotation is based on Chamnongpol et al. 2002 which studied Fe(III)-mediated toxicity and the PmrA/PmrB system. The study showed that the PmrA/PmrB system (which regulates the arn operon including arnF) is important for Fe(III) resistance. However, arnF's connection to iron response is indirect -- iron activates BasS/BasR (PmrA/PmrB homolog), which induces the arn operon, leading to lipid A modification. ArnF itself does not sense or directly respond to iron; it is merely transcriptionally induced as part of a regulon. The IGI evidence code suggests genetic interaction, but the gene's role is downstream in the effector pathway rather than in iron sensing or response per se. Reason: The 'response to iron(III) ion' annotation conflates transcriptional regulation with gene function. ArnF is a lipid flippase whose expression happens to be induced by iron via the BasS-BasR two-component system. The gene does not directly sense, bind, or respond to iron. Many genes in stress-induced regulons would receive inappropriate 'response to X' annotations if mere transcriptional induction were sufficient. The PMID:12139617 study focused on PmrA/PmrB-dependent Fe(III) resistance mechanisms but did not specifically demonstrate a direct role for arnF in iron response. The connection is via operon co-regulation, not direct function. Supporting Evidence: PMID:12139617 Fe(III) exerts its microbicidal activity by a mechanism that is oxygen independent and different from that mediated by Fe(II) PMID:15322361 the BasS-BasR system is essential for this iron-dependent induction of yfbE |
| GO:1901264 carbohydrate derivative transport | IMP PMID:17928292 An undecaprenyl phosphate-aminoarabinose flippase required f... | ACCEPT | Summary: The arnF deletion mutant shows 4-5 fold reduced accessibility of undecaprenyl phosphate-alpha-L-Ara4N to membrane-impermeable sulfo-NHS-biotin labeling, while total levels of the substrate are unchanged [PMID:17928292]. This demonstrates that ArnF is required for transport of this carbohydrate derivative (L-Ara4N-linked lipid) across the inner membrane. The substrate undecaprenyl phosphate-alpha-L-Ara4N contains L-Ara4N (a carbohydrate derivative), making this term appropriate. Reason: This IMP annotation accurately describes the biological process. The sulfo-NHS-biotin accessibility assay in Yan et al. 2007 provides direct evidence that ArnF is involved in transporting the carbohydrate derivative undecaprenyl phosphate-alpha-L-Ara4N across the membrane. This is a core process for ArnF. Supporting Evidence: PMID:17928292 At the 4-h time point, there was a 4-5-fold reduction in ratio of the monoisotopic peak areas for biotinylated undecaprenyl phosphate-alpha-L-Ara4N compared with unmodified undecaprenyl phosphate-alpha-L-Ara4N PMID:17928292 undecaprenyl phosphate-alpha-L-Ara4N levels were the same or slightly higher in the mutants than in the parent, indicating that appropriate amounts of the prenol lipid donor of the L-Ara4N moiety are still synthesized in these mutants |
| GO:1901505 carbohydrate derivative transmembrane transporter activity | IMP PMID:17928292 An undecaprenyl phosphate-aminoarabinose flippase required f... | ACCEPT | Summary: Experimental evidence from the sulfo-NHS-biotin accessibility assay demonstrates that ArnF is required for translocating undecaprenyl phosphate-alpha-L-Ara4N (a carbohydrate derivative) across the inner membrane [PMID:17928292]. The arnF mutant shows reduced periplasmic accessibility of the substrate while total levels remain unchanged, consistent with impaired transporter function. Complementation with pWSK29-PmrM restores polymyxin resistance, confirming the specific role of ArnF [PMID:17928292]. Reason: This MF annotation is well-supported by the experimental data in Yan et al. 2007. The sulfo-NHS-biotin assay demonstrates transporter activity for a carbohydrate derivative substrate. The complementation experiment confirms gene-specific function. This represents a core molecular function of ArnF. Supporting Evidence: PMID:17928292 The data imply that both PmrL and PmrM are involved in the translocation of undecaprenyl phosphate-alpha-LAra4N across the inner membrane PMID:17928292 polymyxin resistance was recovered in the pmrM deletion mutant AY101 by transforming with pWSK29-PmrM |
| GO:0005886 plasma membrane | IDA PMID:15919996 Global topology analysis of the Escherichia coli inner membr... | ACCEPT | Summary: Daley et al. 2005 performed a global topology analysis of the E. coli inner membrane proteome using C-terminal GFP/PhoA fusions. ArnF (YfbJ) was among the 601 inner membrane proteins characterized, with C-terminal tagging establishing its inner membrane localization [PMID:15919996]. In bacterial GO annotation, the inner membrane is annotated as plasma membrane (GO:0005886). Reason: This IDA annotation is based on direct experimental evidence from GFP/PhoA topology analysis. The C-terminal fusion approach provides reliable evidence for inner membrane localization. This is the strongest evidence for the cellular component annotation and represents a core localization for ArnF. Supporting Evidence: PMID:15919996 Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins |
| GO:0010041 response to iron(III) ion | IEP PMID:15322361 A Genome-wide view of the Escherichia coli BasS-BasR two-com... | MARK AS OVER ANNOTATED | Summary: Hagiwara et al. 2004 used genome-wide transcriptome profiling to show that the yfbE operon (now arn operon, including arnF) is induced in response to external iron via the BasS-BasR two-component system [PMID:15322361]. The IEP evidence code (Inferred from Expression Pattern) is technically appropriate for a gene whose expression changes in response to iron. However, as with the IGI annotation above, this annotation conflates transcriptional regulation with direct functional involvement in iron response. ArnF's function is lipid flipping, not iron sensing. Reason: The IEP evidence code is technically appropriate -- arnF expression is induced by iron via the BasS-BasR system. However, annotating a gene to 'response to iron(III) ion' based solely on transcriptional induction conflates regulation with function. ArnF is a flippase that translocates undecaprenyl phosphate-alpha-L-Ara4N; it does not participate in iron sensing, binding, or detoxification. Many genes in iron-responsive regulons (e.g., house-keeping genes in Fur regulon) would be similarly over-annotated. The annotation is not wrong in the strict GO sense (the protein is produced in response to iron), but it does not represent a core function and is misleading about ArnF's actual role. Supporting Evidence: PMID:15322361 the BasS-BasR system is essential for this iron-dependent induction of yfbE PMID:15322361 the hypothetical yfbE operon that appears to be implicated in the modification of lipopolysaccharides is regulated at the level of transcription in response to external iron |
| GO:0140303 intramembrane lipid transporter activity | IMP PMID:17928292 An undecaprenyl phosphate-aminoarabinose flippase required f... | NEW | Summary: ArnF functions as a subunit of a flippase that translocates undecaprenyl phosphate-alpha-L-Ara4N from the cytoplasmic to the periplasmic leaflet of the inner membrane [PMID:17928292]. This is specifically an intramembrane lipid transport event -- the lipid-linked substrate is moved from one leaflet to the other within the same membrane, not transported across the membrane into solution. GO:0140303 (intramembrane lipid transporter activity) precisely captures this function. Reason: This term is more specific and accurate for ArnF's molecular function than the existing GO:0022857 (transmembrane transporter activity) or GO:1901505 (carbohydrate derivative transmembrane transporter activity). The sulfo-NHS-biotin accessibility assay in Yan et al. 2007 directly demonstrates that ArnF is required for moving undecaprenyl phosphate-alpha-L-Ara4N to the periplasmic face of the inner membrane -- an intramembrane lipid translocation event. This should be considered a core MF annotation for ArnF. Supporting Evidence: PMID:17928292 PmrL and PmrM could specifically function to flip undecaprenyl phosphate-alpha-L-Ara4N from the cytosolic to the periplasmic side of the inner membrane, possibly functioning as a heterodimer PMID:17928292 At the 4-h time point, there was a 4-5-fold reduction in ratio of the monoisotopic peak areas for biotinylated undecaprenyl phosphate-alpha-L-Ara4N compared with unmodified undecaprenyl phosphate-alpha-L-Ara4N file:ECOLI/arnF/arnF-deep-research-falcon.md ArnE/ArnF are placed as the flipping step between donor synthesis (ArnB/C/A/D) and transfer to lipid A (ArnT) in the L-Ara4N modification pathway |
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