MlcD is a calmodulin-like myosin light chain that serves as the dedicated light chain for the class I myosin MyoD (Myo1D) in Dictyostelium discoideum. It contains four EF-hand motifs but only retains low-affinity calcium binding (Kd ~52 uM) due to mutations in key Ca2+-coordinating residues of EF-hands 2-4, and thus cannot function as a physiological calcium sensor. Two copies of MlcD bind the MyoD IQ motifs in a calcium-insensitive manner, forming the regulatory light chain component of the Myo1D complex. Through its association with MyoD, MlcD participates in membrane dynamics at macropinocytic cups and actin waves.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005509 calcium ion binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference of calcium ion binding is partially supported but misleading. MlcD retains one functional EF-hand with low-affinity calcium binding (Kd 52 uM), well above physiological levels. The protein cannot function as a sensor of physiological calcium, so this annotation overstates the functional relevance. Reason: MlcD does bind calcium but with very low affinity that is not physiologically relevant. The IBA inference from calmodulin family members with high-affinity calcium binding is misleading for this divergent paralog. The annotation is technically correct but not representative of the core function. Supporting Evidence: PMID:12826013 yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+ PMID:12826013 The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+ |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Cytoplasmic localization is correct and well-supported by direct evidence from the same gene product. Reason: MlcD is a cytoplasmic protein as shown by direct experimental evidence, consistent with its role as a myosin light chain that operates in the cytoplasm. Supporting Evidence: PMID:12826013 FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD |
| GO:0030234 enzyme regulator activity | IBA GO_REF:0000033 | REMOVE | Summary: This IBA annotation from the calmodulin family is questionable for MlcD. While calmodulin regulates numerous enzymes, MlcD has diverged substantially and specifically associates only with MyoD. There is no direct evidence that MlcD regulates enzyme activity per se; it functions as a structural light chain of a motor complex rather than as an enzyme regulator. Reason: MlcD is a dedicated structural light chain for MyoD, not a general enzyme regulator. Unlike calmodulin, which activates kinases and phosphatases, MlcD binds only MyoD in a calcium-insensitive manner. The IBA inference from calmodulin family members does not transfer to this divergent paralog that has lost both high-affinity calcium binding and promiscuous target engagement. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000549861 · PANTHER node for the calmodulin family SUPPORTS SOURCE BUT NOT TARGET Enzyme-regulator activity reflects calmodulin's general regulatory role; MlcD is a structural light chain for MyoD and is not a general enzyme regulator UniProtKB:P62203 · human calmodulin (CALM) SUPPORTS SOURCE BUT NOT TARGET Calmodulin regulates many target enzymes; this generic regulator role does not transfer to the dedicated myosin light chain Supporting Evidence: PMID:12826013 FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC PMID:12826013 The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+ |
| GO:0000226 microtubule cytoskeleton organization | IBA GO_REF:0000033 | REMOVE | Summary: This IBA annotation transfers microtubule-related functions from calmodulin orthologs. There is no evidence that MlcD or its partner MyoD are involved in microtubule cytoskeleton organization. MyoD/Myo1D is an actin-based motor that functions at membranes and actin waves. Reason: MlcD is a dedicated myosin I light chain with no known connection to microtubule biology. All experimental evidence places MlcD/MyoD in the context of actin-based structures (macropinocytic cups, actin waves). The IBA inference from calmodulin, which does participate in microtubule biology through different targets, does not transfer to this divergent paralog. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000549862 · PANTHER node for the calmodulin / EF-hand light-chain family SUPPORTS SOURCE BUT NOT TARGET Microtubule-organization roles derive from multifunctional calmodulin-family members; MlcD is a dedicated myosin I light chain with no microtubule connection FB:FBgn0000253 · Drosophila calmodulin (Cam) SUPPORTS SOURCE BUT NOT TARGET Multifunctional calmodulin source; its cytoskeletal roles do not transfer to a dedicated myosin light chain Supporting Evidence: PMID:31774725 Myo1D localized, like short-tail Myo1E and Myo1F, in the PIP3-rich region encircled by the actin wave PMID:12826013 FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC |
| GO:0005509 calcium ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO annotation from the EF-hand domain (IPR002048). While MlcD does contain EF-hand domains and retains low-affinity calcium binding, this annotation overstates functional importance since the binding is not physiologically relevant. Reason: The InterPro2GO inference from EF-hand domains is technically correct (MlcD does bind calcium) but functionally misleading. Three of four EF-hands have degenerate calcium-coordinating residues, and the measured Kd of 52 uM is far above physiological calcium concentrations. This is not a core function. Supporting Evidence: PMID:12826013 EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding PMID:12826013 The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+ |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Correct annotation based on UniProt subcellular location. MlcD is a cytoplasmic protein. Reason: Cytoplasmic localization is well established for MlcD from direct experimental evidence. Supporting Evidence: PMID:12826013 FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD |
| GO:0005509 calcium ion binding | IDA PMID:12826013 The Dictyostelium class I myosin, MyoD, contains a novel lig... | KEEP AS NON CORE | Summary: Direct experimental evidence demonstrates calcium binding by MlcD via electrospray ionization MS and tryptophan fluorescence quenching. However, the binding is low affinity (Kd 52 uM), so while the annotation is experimentally supported, it does not represent the core evolved function. Reason: The IDA evidence from De La Roche et al. 2003 is solid - MlcD does bind calcium as demonstrated by ESI-MS and fluorescence spectroscopy. However, the authors themselves conclude the binding is not physiologically relevant. This is a vestigial capacity from calmodulin ancestry, not a core function. Supporting Evidence: PMID:12826013 Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site PMID:12826013 yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+ PMID:12826013 The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+ |
| GO:0016459 myosin complex | IDA PMID:12826013 The Dictyostelium class I myosin, MyoD, contains a novel lig... | MODIFY | Summary: MlcD is part of the MyoD (Myo1D) myosin complex. This is a core annotation. The more specific term GO:0045160 myosin I complex would be more precise since MyoD is a class I myosin. Reason: MlcD co-purifies with MyoD as part of a myosin I complex (two copies of MlcD per MyoD heavy chain). The annotation is correct but the more specific child term myosin I complex is more appropriate since MyoD is definitively a class I myosin. Proposed replacements: myosin I complex Supporting Evidence: PMID:12826013 MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain PMID:21671662 Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD) |
| GO:0032036 myosin heavy chain binding | IPI PMID:12826013 The Dictyostelium class I myosin, MyoD, contains a novel lig... | MODIFY | Summary: MlcD specifically binds the MyoD heavy chain at its IQ motifs. Since MyoD is a class I myosin, the more specific term GO:0032037 myosin I heavy chain binding is more appropriate. Reason: MlcD binds specifically to MyoD (a class I myosin) heavy chain IQ motifs. The general term myosin heavy chain binding is correct but the child term myosin I heavy chain binding more precisely captures the specificity of this interaction. Proposed replacements: myosin I heavy chain binding Supporting Evidence: PMID:12826013 Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs PMID:12826013 FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC |
| GO:0062201 actin wave | IDA PMID:31774725 Basic-hydrophobic sites are localized in conserved positions... | ACCEPT | Summary: MlcD localizes to actin waves as part of the Myo1D complex. Specifically, Myo1D localizes to the PIP3-rich region encircled by the actin wave, not the actin wave crest itself. This is a valid localization annotation reflecting the behavior of the Myo1D complex. Reason: Brzeska et al. 2019 directly demonstrated Myo1D (and hence its light chain MlcD) localizing to actin wave structures, specifically in the PIP3-rich region. This is a core localization for the Myo1D motor complex. Supporting Evidence: PMID:31774725 Myo1D localized, like short-tail Myo1E and Myo1F, in the PIP3-rich region encircled by the actin wave |
| GO:0070687 macropinocytic cup cytoskeleton | IDA PMID:31774725 Basic-hydrophobic sites are localized in conserved positions... | ACCEPT | Summary: Myo1D localizes to macropinocytic cups, and MlcD as its light chain would be present there. The annotation to macropinocytic cup cytoskeleton is well supported by the localization data. Reason: Brzeska et al. 2019 showed that Myo1D localizes to macropinocytic protrusions, cups, and freshly internalized vesicles. As the obligate light chain, MlcD is present at these sites. Supporting Evidence: PMID:31774725 Myo1D is also found in pinocytic protrusions and cups, and freshly internalized vesicles file:DICDI/mlcD/mlcD-deep-research-bioreason-sft.md Its recruitment to large endocytic structures is consistent with GO:0070685 macropinocytic cup |
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