MlaB (YrbB) is a small (97-residue) cytoplasmic protein consisting of a single STAS (sulfate transporter and anti-sigma factor antagonist) domain, encoded as the last gene of the mlaFEDCB operon of the Mla (maintenance of lipid asymmetry) intermembrane phospholipid transport system. Two MlaB subunits are stoichiometric components of the inner-membrane MlaFEDB ABC transporter complex, each bound to the side of the helical subdomain of one MlaF ATPase on the cytoplasmic face of the inner membrane, far from the ATP sites and away from the MlaE/MlaD lipid pathway. MlaB binds and stabilizes MlaF: without MlaB, MlaF cannot be produced in useful amounts and does not assemble with MlaE and MlaD. A T52A substitution at the conserved STAS Ser/Thr position, which lies at the MlaF interface, leaves complex assembly intact but abolishes complex ATPase activity. Over-expression of MlaF suppresses the mlaB deletion, suggesting that the main role of MlaB is to keep MlaF stable and available for MlaFEDB assembly; a regulatory role (for example by phosphorylation of Thr52) has been proposed but not demonstrated. Loss of MlaB causes outer-membrane lipid asymmetry defects and SDS-EDTA sensitivity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:32602838 Structure of MlaFB uncovers novel mechanisms of ABC transpor... | REMOVE | Summary: MlaB binds the MlaF nucleotide-binding domain (MlaFB crystal structure, pull-downs). Reason: The interaction is well established, but bare protein binding is uninformative under the protein-binding policy. Its functional consequence, stabilization of MlaF and its assembly into MlaFEDB, is captured by the NEW protein stabilization annotation and the complex membership term (GO:0043190). Removal does not dispute the interaction. Supporting Evidence: PMID:32602838 MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. |
| GO:0005515 protein binding | IPI PMID:33199922 Structural insights into outer membrane asymmetry maintenanc... | REMOVE | Summary: MlaB-MlaF contact within the cryo-EM MlaFEDB structure. Reason: Bare protein binding is uninformative; the MlaF contact is a subunit interaction within MlaFEDB, represented by complex membership (GO:0043190) and the protein stabilization process. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: MlaB is a soluble cytoplasmic STAS-domain protein. Reason: MlaB has no signal peptide or transmembrane segment and binds the cytoplasmic MlaF subunits of MlaFEDB. Supporting Evidence: PMID:33236984 Outside this ABC transporter core, MlaFEDB contains additional subunits not found in other ABC transporters: MlaD on the periplasmic side of the IM, and MlaB in the cytoplasm. |
| GO:0005829 cytosol | IDA PMID:17309111 Comparison of SDS- and methanol-assisted protein solubilizat... | ACCEPT | Summary: Proteomic detection reported by EcoCyc as cytosolic localization. Reason: Abstract-only cache, so the specific observation cannot be checked, but a soluble cytoplasmic location is consistent with all structural data for MlaB; defer to the curator. Supporting Evidence: PMID:33236984 Outside this ABC transporter core, MlaFEDB contains additional subunits not found in other ABC transporters: MlaD on the periplasmic side of the IM, and MlaB in the cytoplasm. |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Detection of MlaB in the E. coli cytosolic proteome. Reason: MlaB is not named in the cached main text (presumably in the supplementary protein lists), but cytosolic detection agrees with MlaB being a soluble cytoplasmic protein; defer to the curator. Supporting Evidence: PMID:33236984 Outside this ABC transporter core, MlaFEDB contains additional subunits not found in other ABC transporters: MlaD on the periplasmic side of the IM, and MlaB in the cytoplasm. |
| GO:0006974 DNA damage response | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT propagates DNA damage response to the gammaproteobacterial MlaB node, seeded only by the E. coli mlaB transposon-mutant phenotype. Reason: The only seed is the yrbB::Tn5 hyperlethality phenotype (PMID:20128927), an indirect consequence of an outer-envelope defect. Nothing places MlaB in a DNA-damage sensing or repair pathway, so propagating involvement in the DNA damage response across the MlaB family overstates a pleiotropic phenotype. The PTHR35849 family review assesses this node as WRONG_NODE for the same reason. MARK_AS_OVER_ANNOTATED rather than REMOVE because the seed phenotype itself is real and verified in the full text, and this row's sole seed is the gene's own IMP row below, so the two rows make one claim and carry the same action. What is wrong is the process assignment, not the observation. Propagation Review Root cause: SOURCE WEAK OR INFERRED Failure modes: ROLE CONFLATION SOURCE EVIDENCE WEAK Sources checked: PANTHER:PTN002185077 · PTN002185077 SOURCE WEAK OR INFERRED Sole seed is P64602's own acts_upstream_of_or_within IMP from a Tn5 hyperlethality screen; an indirect envelope phenotype, not participation in the DNA damage response, was converted into an involved_in IBD. Supporting Evidence: PMID:20128927 Eight of the mutant genes (yadC, rfbX, rfbC, ycdO, yrbB, ybdA, emrK, and emrY) were annotated in Genbank as outer membrane proteins or proteins whose function is related to the outer membrane. |
| GO:0006974 DNA damage response | IMP PMID:20128927 Escherichia coli genes that reduce the lethal effects of str... | MARK AS OVER ANNOTATED | Summary: yrbB::Tn5 mutants are more readily killed by nalidixic acid, mitomycin C and UV. Reason: The hyperlethality phenotype is real (full text verified, complemented), but the authors group yrbB with outer-membrane-related genes and offer no mechanism linking MlaB to a DNA-damage response; the effect is most plausibly an indirect consequence of compromised envelope homeostasis in Mla mutants. A DNA damage response process term overstates the role of MlaB. Supporting Evidence: PMID:20128927 When the DNA damaging agent mitomycin C was tested, all of the mutants were more readily killed than wild-type cells PMID:20128927 Eight of the mutant genes (yadC, rfbX, rfbC, ycdO, yrbB, ybdA, emrK, and emrY) were annotated in Genbank as outer membrane proteins or proteins whose function is related to the outer membrane. |
| GO:0015914 phospholipid transport | NAS PMID:19383799 An ABC transport system that maintains lipid asymmetry in th... | MODIFY | Summary: The founding Mla paper proposes an intermembrane phospholipid trafficking pathway including MlaB. Reason: Correct but general; the specific supported process is intermembrane phospholipid transfer (GO:0120010), which MlaB already carries by IBA and IDA. Proposed replacements: intermembrane phospholipid transfer Supporting Evidence: PMID:19383799 We propose that the Mla pathway constitutes a bacterial intermembrane PL trafficking system. |
| GO:0016020 membrane | NAS PMID:29358647 Unanticipated functional diversity among the TatA-type compo... | MODIFY | Summary: MlaB is associated with the inner membrane only through MlaF in MlaFEDB; the cited paper (TatA-type Tat translocase components) does not concern Mla. Reason: MlaB is not itself a membrane protein, but as a stoichiometric MlaFEDB subunit bound to MlaF it sits at the cytoplasmic face of the inner membrane, so GO:0031234 extrinsic component of cytoplasmic side of plasma membrane is the accurate refinement. PMID:29358647 has no mention of Mla proteins in its full text and does not support the row. Proposed replacements: extrinsic component of cytoplasmic side of plasma membrane Supporting Evidence: PMID:33236984 On the cytoplasmic side, each of the MlaF ATPase subunits is bound to MlaB |
| GO:0046677 response to antibiotic | IMP PMID:20128927 Escherichia coli genes that reduce the lethal effects of str... | KEEP AS NON CORE | Summary: The yrbB::Tn5 mutant is hyperlethal to nalidixic acid (complemented by plasmid yrbB) without a change in MIC. Reason: Verified in the full text; the antibiotic lethality phenotype is the primary screen readout and was complemented, so the acts_upstream_of_or_within annotation is supported. It is an indirect consequence of envelope homeostasis defects, not a core MlaB function. Supporting Evidence: PMID:20128927 These data indicate that the yrbB mutation does not change the permeablility to most compounds, which is consistent with our finding that the yrbB mutation did not change the bacteriostatic effects of nalidixic acid. PMID:20128927 In each case, complementation was observed |
| GO:0120010 intermembrane phospholipid transfer | IBA GO_REF:0000033 | ACCEPT | Summary: MlaB is a required subunit of the MlaFEDB complex that drives the inner-membrane step of Mla intermembrane phospholipid transfer. Reason: MlaB contributes structurally to the transporter by stabilizing MlaF and enabling MlaFB assembly with MlaED; mlaB mutants have the Mla lipid-asymmetry phenotype. The gammaproteobacterial MlaB node placement is appropriate. Supporting Evidence: PMID:27529189 Finally, we establish that MlaB is necessary for both the assembly and activity of the ABC transporter, likely by modulating MlaF structure and stability. |
| GO:0120010 intermembrane phospholipid transfer | IDA PMID:33199922 Structural insights into outer membrane asymmetry maintenanc... | ACCEPT | Summary: Reconstituted MlaFEDB (including MlaB) transports phospholipid between proteoliposomes with MlaC and MlaA-OmpF. Reason: Abstract-only cache, but the abstract describes the reconstituted transport system containing MlaFEDB; consistent with the full-text evidence that MlaB is required for MlaFEDB assembly and activity. Defer to the curator for assay details. Supporting Evidence: PMID:33199922 establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA-OmpF to monitor the transport direction of phospholipids |
| GO:0120014 phospholipid transfer activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT asserts that MlaB enables phospholipid transfer activity; MlaB is a cytoplasmic STAS subunit that does not contact lipid. Reason: GO:0120014 describes carrying a phospholipid between membranes in a hydrophobic pocket. In MlaFEDB the lipid pathway runs through MlaE and the MlaD ring, the ATP that drives it is hydrolyzed by MlaF, and MlaB binds the far side of MlaF in the cytoplasm. MlaF over-expression suppresses the mlaB deletion, so MlaB is not intrinsically needed for the transfer chemistry. MlaB at most contributes to the complex-level activity (recorded as contributes_to in core_functions); enabling the activity overstates its role. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION Sources checked: PANTHER:PTN002185077 · PTN002185077 SUPPORTS SOURCE BUT NOT TARGET Sole seed is P64602's own IDA from a reconstituted MlaFEDB assay, which measures the whole complex. Propagating enables to the non-catalytic, non-lipid-binding STAS subunit conflates complex-level activity with subunit activity; contributes_to would be the appropriate relation. Supporting Evidence: PMID:33236984 A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. PMID:32602838 This suggests that MlaB may have no essential function in Mla lipid transport apart from its role in promoting MlaF stability and/or association with the MlaFEDB complex. |
| GO:0120014 phospholipid transfer activity | IDA PMID:33199922 Structural insights into outer membrane asymmetry maintenanc... | MARK AS OVER ANNOTATED | Summary: Phospholipid transfer measured with reconstituted MlaFEDB, attributed to each subunit including MlaB. Reason: The assay measures the activity of the whole MlaFEDB complex. MlaB does not bind or move phospholipid; its documented role is binding and stabilizing MlaF. The annotation is defensible only as contributes_to (see core_functions), not as an activity enabled by MlaB itself. Supporting Evidence: PMID:33199922 establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA-OmpF to monitor the transport direction of phospholipids PMID:32602838 These data suggest that MlaB stabilizes MlaF, thus allowing the assembly of MlaFEDB transporters |
| GO:1990531 phospholipid-translocating ATPase complex | IBA GO_REF:0000033 | MODIFY | Summary: MlaB is a stoichiometric subunit of MlaFEDB, but GO:1990531 is defined as a P-type (P4) ATPase flippase complex. Reason: Complex membership is correct, but GO:1990531 is defined as "A protein complex that functions as a phospholipid-translocating P-Type ATPase" (synonyms P4-ATPase complex, DRS2-CDC50 complex) and is not classified under ABC transporter complexes. MlaFEDB is an ABC transporter, so GO:0043190 ATP-binding cassette (ABC) transporter complex is the correct term. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN002185077 · PTN002185077 SUPPORTS TRANSFER Seeded by P64602; MlaFEDB membership transfers across the gammaproteobacterial MlaB clade, but the asserted term is the P-type ATPase flippase complex rather than the ABC transporter complex. Proposed replacements: ATP-binding cassette (ABC) transporter complex Supporting Evidence: PMID:33236984 Outside this ABC transporter core, MlaFEDB contains additional subunits not found in other ABC transporters: MlaD on the periplasmic side of the IM, and MlaB in the cytoplasm. |
| GO:1990531 phospholipid-translocating ATPase complex | IPI PMID:27529189 Defining key roles for auxiliary proteins in an ABC transpor... | MODIFY | Summary: Affinity purification shows MlaB co-purifies in a stable MlaFEDB complex; the term chosen is the P-type ATPase flippase complex. Reason: The experiment robustly supports MlaB membership in the MlaFEDB ABC transporter complex, but GO:1990531 is defined as a P-type ATPase complex. Replace with GO:0043190 ATP-binding cassette (ABC) transporter complex. Proposed replacements: ATP-binding cassette (ABC) transporter complex Supporting Evidence: PMID:27529189 We show that MlaF, MlaE, MlaD and MlaB interact specifically with each other to form a stable complex. |
| GO:0050821 protein stabilization | IDA PMID:32602838 Structure of MlaFB uncovers novel mechanisms of ABC transpor... | NEW | Summary: MlaB binds and stabilizes the MlaF ATPase, allowing MlaF to accumulate and assemble with MlaE and MlaD. Reason: MlaB itself performs the stabilization by binding the MlaF NBD. MlaF cannot be expressed and purified alone but is obtained in good yield when co-expressed with MlaB; without MlaB, MlaF does not stably associate with MlaE/MlaD; and MlaF over-expression bypasses the mlaB deletion, indicating that MlaF stabilization is the principal MlaB activity. Thong et al. independently found reduced MlaF levels without MlaB. Supporting Evidence: PMID:32602838 In contrast, we were unable to express and purify significant amounts of MlaF alone with either an N-terminal or C-terminal His tag. PMID:32602838 These data suggest that MlaB stabilizes MlaF, thus allowing the assembly of MlaFEDB transporters PMID:32602838 Thus, over-expression of MlaF can fully suppress the defect of the mlaB knock-out strain. PMID:27529189 In fact, the affinity-enriched levels of MlaF-His appear to be reduced in the absence of MlaB, suggesting that MlaB modulates the stability of MlaF. |
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Download this section (compressed HTML)Q: Is MlaB Thr52 phosphorylated in vivo, and if so by which kinase and under what envelope-stress conditions?
Q: Does the T52A defect in ATPase activity reflect loss of a regulatory signal or a perturbed MlaF-MlaB interface that destabilizes the MlaF dimer?
Experiment: Phosphoproteomic detection of MlaB Thr52 across growth phases and envelope stresses, combined with phosphomimetic (T52D/E) and non-phosphorylatable alleles assayed for MlaF levels, MlaFEDB ATPase activity and SDS-EDTA resistance.
Type: phosphoproteomics and site-directed mutagenesis
Experiment: Measure MlaF half-life in vivo by translational shut-off chase in wild type, mlaB deletion and mlaB T52A strains.
Type: protein stability assay
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether MlaB has any role beyond stabilizing MlaF, such as regulation of MlaF ATPase activity through phosphorylation of the conserved STAS Thr52, is unknown; T52A abolishes complex ATPase activity, yet MlaF over-expression bypasses the mlaB deletion.
OPEN BIOLOGY
Significance: Determines whether MlaB is purely a structural/stabilizing subunit or a regulatory input that tunes Mla transport flux.
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