Existing Annotations Review

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

Core Functions

MlcD serves as the dedicated regulatory light chain subunit of the Myo1D (MyoD) class I myosin complex. Two copies of MlcD bind the MyoD heavy chain IQ motifs in a calcium-insensitive manner, providing structural stabilization of the motor complex neck region.

Molecular Function:

myosin I heavy chain binding

Cellular Locations:

cytoplasm actin wave macropinocytic cup cytoskeleton

In Complex:

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:12826013
Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs
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)

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

PMID:12826013

The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.

MlcD is a 16 kDa calmodulin-like protein that co-purifies with MyoD as two copies per heavy chain.

"MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain"
MlcD has four EF-hands but only retains low-affinity calcium binding due to degenerate calcium-coordinating residues in EF-hands 2-4.

"MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding"
MlcD binds calcium with a Kd of 52 uM, far above physiological calcium concentrations.

"yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+"
MlcD cannot function as a physiological calcium sensor.

"The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+"
MlcD binding to MyoD is calcium-insensitive.

"Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs"
MlcD specifically associates with MyoD and not with MyoB or MyoC.

"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:16415352

Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin.

MlcB is a distinct light chain for MyoB, confirming that each long-tailed myosin I has a unique light chain partner.

"FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD"

PMID:21671662

Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes.

Each long-tailed Dictyostelium myosin I has a unique light chain, while short-tailed ones use calmodulin.

"Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain"
Light chain diversity contributes to functional specialization of myosin I isoforms.

"The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme"

PMID:31774725

Basic-hydrophobic sites are localized in conserved positions inside and outside of PH domains and affect localization of Dictyostelium myosin 1s.

Myo1D localizes to the PIP3-rich region inside actin waves, distinct from Myo1B which colocalizes with the actin wave itself.

"Myo1D localized, like short-tail Myo1E and Myo1F, in the PIP3-rich region encircled by the actin wave"
Myo1D localizes to macropinocytic cups and freshly internalized vesicles.

"Myo1D is also found in pinocytic protrusions and cups, and freshly internalized vesicles"

Deep Research

Bioreason Pro

(mlcD-deep-research-bioreason-sft.md)

Thinking Trace Bioreason Pro SFT

Thinking Trace

The architecture begins with IPR050230 (Calmodulin/Myosin light chain/Troponin C-like family, residues 2–146), which defines a compact, helical EF-hand calcium sensor specialized for binding target peptides such as IQ motifs on myosins. This family scaffold is refined by IPR011992 (EF-hand domain pair homologous superfamily, residues 5–144), indicating two EF-hand pairs arranged N- and C-terminally. The N-terminal pair is marked by IPR002048 EF-hand domains at residues 8–43, 12–40, and 14–60, with IPR018247 specifying the canonical Ca2+-coordinating loop at residues 21–33 within this first pair. The C-terminal pair is demarcated by IPR002048 domains at residues 79–114 and 81–144, with overlapping confidence windows (83–111 and 83–144) that emphasize a functional Ca2+-binding site in the second lobe. This ordered layout—two Ca2+-binding pairs within a calmodulin-like fold—causes Ca2+-dependent conformational switching that exposes hydrophobic pockets for target engagement.

This EF-hand architecture directly encodes GO:0005509 calcium ion binding as the primary molecular function. The calmodulin/myosin light chain-like family context predicts high-affinity recognition of IQ motifs on myosin heavy chains, supporting GO:0032036 myosin heavy chain binding. In this protein, the paired EF-hands act as a Ca2+ sensor that toggles between low- and high-affinity states for myosin II regulatory segments, thereby modulating motor activity.

Calcium-gated control of myosin II provides a mechanistic route to multiple cellular processes. By binding the myosin II heavy chain, the protein can tune filament assembly and spacing at the cleavage furrow, accounting for GO:0043520 regulation of myosin II filament assembly and GO:0000281 mitotic cytokinesis. Targeting to the division plane is consistent with GO:0071976 protein localization to cell division site. The same Ca2+-dependent exposure of target pockets can regulate interactions with actin regulators, explaining GO:0030041 actin filament polymerization and GO:0030837 negative regulation of actin filament polymerization, as different target states promote or restrain filament growth depending on Ca2+ levels. In endocytic and phagocytic remodeling, Ca2+ spikes recruit this sensor to the cortex to coordinate myosin II contractility and actin assembly, aligning with GO:0006911 phagocytosis, engulfment and GO:0006907 pinocytosis. Rapid Ca2+-triggered assembly at membrane lesions leverages the same mechanism for GO:0001778 plasma membrane repair. By modulating the activity of myosin II and associated enzymes, the protein exerts GO:0043462 regulation of ATP-dependent activity. Its Ca2+-dependent binding to kinases, phosphatases, and small GTPase regulators provides a route to GO:0043087 regulation of GTPase activity and GO:0032092 positive regulation of protein binding. In Dictyostelium development, these cytoskeletal and signaling effects influence multicellular aggregation dynamics (GO:0031152 aggregation involved in sorocarp development) and can restrain proliferative cycles, supporting GO:1903665 negative regulation of asexual reproduction. Nuclear actomyosin and envelope mechanics suggest a role in GO:0006997 nucleus organization.

The EF-hand sensor lacks transmembrane segments and is optimized for dynamic association with actomyosin assemblies, placing it in the GO:0016459 myosin complex and at the GO:0030864 cortical actin cytoskeleton. Its recruitment to large endocytic structures is consistent with GO:0070685 macropinocytic cup. These localizations follow directly from Ca2+-induced exposure of myosin-binding pockets and affinity for IQ motifs on myosin II at the cortex and division site.

A coherent mechanistic model emerges: Ca2+ binding to the N-terminal EF-hands primes the sensor to engage myosin II heavy chain IQ motifs, while the C-terminal EF-hands stabilize the complex and tune affinity. This engagement alters myosin II filament assembly and spacing, thereby controlling contractility and actin polymerization at the cleavage furrow, during macropinocytosis, phagocytosis, and membrane repair. Through Ca2+-dependent docking, the protein likely associates with ROCO-family kinases (roco11, roco6, roco5, roco8) to couple cytoskeletal state to kinase signaling; with the serine/threonine-protein phosphatase 2B catalytic subunit to reset phosphorylation states in a Ca2+-dependent manner; with an inositol 1,4,5-trisphosphate receptor-like protein A to integrate Ca2+ release dynamics; and with the Ras GTPase-activating-like protein gapA to coordinate small GTPase cycles. Together, these interactions position the protein as a Ca2+-sensor that orchestrates myosin II–driven mechanics and signaling at the cortex and division site.

## Functional Summary

A calmodulin-like calcium sensor in Dictyostelium that binds calcium to expose target-specific pockets and engage the myosin II heavy chain. By tuning myosin II filament assembly and contractility at the cortex and cleavage furrow, it coordinates actin polymerization dynamics for cytokinesis, macropinocytosis, phagocytic engulfment, and rapid plasma membrane repair. Its calcium-dependent interactions with kinases, phosphatases, and small GTPase regulators integrate cytoskeletal mechanics with signaling, influencing aggregation during development and restraining asexual proliferation. The protein operates within myosin-containing assemblies at the cortical actin cytoskeleton and macropinocytic cups.

## UniProt Summary

Binds calcium. Binds to the myosin II heavy chain.

## InterPro Domains

    - IPR050230: Calmodulin/Myosin light chain/Troponin C-like (family) [2-146]
- IPR011992: EF-hand domain pair (homologous_superfamily) [5-144]
- IPR002048: EF-hand domain (domain) [8-43]
- IPR002048: EF-hand domain (domain) [12-40]
- IPR002048: EF-hand domain (domain) [14-60]
- IPR018247: EF-Hand 1, calcium-binding site (binding_site) [21-33]
- IPR002048: EF-hand domain (domain) [79-114]
- IPR002048: EF-hand domain (domain) [81-144]
- IPR002048: EF-hand domain (domain) [83-111]
- IPR002048: EF-hand domain (domain) [83-144]

## GO Term Predictions

### Molecular Function


### Biological Process


### Cellular Component

OpenScientist

(mlcD-hypotheses/prediction-myosin-cytokinesis/openscientist.md)

mlcD (Q7Z2B8) — Myosin-II / Cytokinesis Prediction Assessment OpenScientist openscientist-autonomous 3 citations 2 artifacts 2026-07-10T03:53:33.149597 citations file

OpenScientist final report (text/html)

OpenScientist final report (application/pdf)

mlcD (Q7Z2B8) — Myosin-II / Cytokinesis Prediction Assessment

Gene: mlcD (UniProt Q7Z2B8, MLCD_DICDI), Dictyostelium discoideum (NCBITaxon:44689)
Focus: computational_prediction — hypothesis prediction-myosin-cytokinesis
Terms tested: GO:0000281 mitotic cytokinesis; GO:0030837 negative regulation of actin filament polymerization; GO:0043520 regulation of myosin II filament assembly; GO:0071976 protein localization to cell division site.
Reference context: doi:10.64898/2026.03.19.712954

Executive Judgment

Verdict: REFUTED (over-annotation / paralog–class misassignment).

BioReason-Pro-SFT predicts myosin-II and cytokinesis regulatory roles for mlcD. Primary
biochemistry and the curated UniProt/dictyBase record independently establish that mlcD is
the dedicated essential light chain of MyoD, a single-headed class I (unconventional) myosin —
not a component of the conventional myosin-II (mhcA) contractile machinery. All four predicted
terms belong to the myosin-II system (heavy chain mhcA plus its regulatory light chain and
essential light chain), whose bipolar filaments drive cytokinesis. Class I myosins are monomeric,
membrane-associated motors that do not self-assemble into filaments, so mlcD cannot mediate or
regulate myosin-II filament assembly or contractile-ring–based cytokinesis.

The most important caveat: I could not access the specific reference doi:10.64898/2026.03.19.712954
programmatically, and I did not run a MyoD-null phenotype search successfully (PubMed queries for
class-I phenotypes returned no hits in this environment). The refutation nonetheless rests on
direct, unambiguous primary evidence (protein sequencing + complex purification) and on the
curated functional record.

Evidence Matrix

Citation	Evidence type	Direction	Claim tested	Key finding	Context	Confidence / limitations
PMID:12826013 (De La Roche, Lee, Côté 2003)	Direct assay (protein sequencing, ESI-MS, ITC, complex purification)	Refutes seed	Is mlcD a myosin-II light chain?	MlcD is a 16 kDa 4-EF-hand protein co-purifying with MyoD, a long-tailed class I myosin; FLAG-MlcD complexes with MyoD but not MyoB/MyoC; low Ca²⁺ affinity (cannot sense physiological Ca²⁺)	D. discoideum, native + FLAG-tagged expression	High. Directly defines class/partner identity.
PMID:21671662 (Crawley et al. 2011)	Interaction / biochemical mapping	Refutes seed	Light-chain assignments across Dictyostelium myosin-I	Each long-tailed myosin-I has a unique light chain: MyoB–MlcB, MyoC–MlcC, MyoD–MlcD; short-tailed MyoA/MyoE use calmodulin	D. discoideum	High. Confirms MyoD–MlcD pairing.
UniProt Q7Z2B8 (MLCD_DICDI)	Database / curated record	Refutes seed	Protein identity, family, oligomeric state	Recommended name "Myosin-ID light chain"; FUNCTION "light chain for myosin-D"; SUBUNIT "Myosin I… Inability to self-assemble into filaments… Interacts with myoD; does not interact with myoB or myoC"	Curated	High (review/database-level, but backed by the primary papers above).
dictyBase GO (via UniProt xrefs)	Database (experimental IDA/IPI)	Qualifies / competing	What processes/locations are experimentally supported?	Curated terms: myosin complex (IDA), myosin heavy chain binding→myoD (IPI), Ca²⁺ binding (IDA), actin wave (IDA), macropinocytic cup cytoskeleton (IDA). None of the 4 predicted myosin-II/cytokinesis terms present	D. discoideum	High. Absence of predicted terms in curated set is informative.
InterPro/PANTHER/Pfam (Q7Z2B8)	Structural/evolutionary	Qualifies	Domain architecture	147 aa, four EF-hands (EF-hands 2–4 degenerate), CALM/Myosin/TropC-like (IPR050230), calmodulin-like — consistent with an EF-hand myosin light chain, not a filament-assembly regulator	Sequence	High.
Iteration-2 NW/BLOSUM62 (this study)	Computational (sequence identity)	Qualifies/refutes	Which clade does MlcD belong to?	MlcD 45.6% id to calmodulin & 45.2% to MlcB (myosin-I LC) vs only 30.8%/31.2% to myosin-II mlcR/mlcE; reproduces published ~44% CaM benchmark	D. discoideum paralogs	Medium-high. Global alignment, single method; concordant with primary data.
PMID:10423462 (Dai et al. 1999)	Mutant phenotype (micropipette aspiration)	Qualifies (class context)	What do Dictyostelium myosin-I motors do?	Amoeboid myosin-I's drive pseudopod formation, macropinocytosis; double mutants lose ~50% cortical tension; required for migration — NOT cytokinesis	D. discoideum myosin-I mutants	Medium (about myosin-I class generally, not MyoD-specific).

GO Curation Implications (leads — require curator verification)

Do NOT add GO:0000281 (mitotic cytokinesis), GO:0030837 (neg. reg. actin filament polymerization),
GO:0043520 (regulation of myosin II filament assembly), or GO:0071976 (protein localization to cell
division site) to mlcD. These are conventional-myosin-II / contractile-ring functions and are
class-inappropriate for a myosin-I light chain.
GO:0043520 specifically: refuted. The correct effectors of Dictyostelium myosin-II filament
assembly are the mhcA heavy chain (regulated by myosin heavy-chain kinases) and its regulatory /
essential light chains — not MlcD. If any myosin light chain warrants GO:0043520, it is the
myosin-II RLC/ELC paralog, not mlcD.
Retain / support instead (MF/CC): MF GO:0032036 myosin heavy chain binding (IPI to myoD) and
the calmodulin-like light-chain role; CC GO:0016459 myosin complex. Prefer these informative terms
over generic "protein binding."
Class-appropriate BP/CC leads (verify against MyoD phenotype literature): class-I myosin
processes at the plasma membrane — macropinocytosis/endocytosis and cortical actin dynamics
(consistent with curated macropinocytic cup cytoskeleton and actin wave localizations).

Mechanistic Scope

Direct molecular function of the gene product: MlcD is a calmodulin-like EF-hand essential light
chain that binds the IQ motifs in the neck of the class I myosin heavy chain MyoD, stabilizing
its lever arm. Its Ca²⁺ affinity is low, so it is not a Ca²⁺ sensor; De La Roche et al. propose it
confers Ca²⁺-insensitive regulatory properties distinguishing MyoD from calmodulin-bearing myosin-I.
The predicted terms describe downstream cellular processes of a different motor system (myosin-II
contractile ring); they are neither the immediate activity of MlcD nor a documented phenotype of it.

Conflicts and Alternatives

Paralog/class confusion (most likely source of the prediction): "mlc*" myosin light-chain genes
form a paralog pool spanning classes. The myosin-II regulatory (mlcR) and essential (mlcE) light
chains legitimately carry cytokinesis/filament-assembly context; a sequence/name-similarity or
frequency-biased transfer plausibly propagated those terms onto mlcD.
Organism specificity: In Dictyostelium, cytokinesis A depends on conventional myosin-II
(mhcA); myosin-I isoforms function in motility, cortical tension, and endocytosis. This reinforces
that mlcD's contribution, if any, is not contractile-ring cytokinesis.
No competing evidence found that MlcD associates with mhcA or the contractile ring.

Knowledge Gaps

MyoD-null / MlcD-null cellular phenotype. Partially addressed: MyoD-specific knockout papers
were not retrievable here, but class-level phenotype data (Dai et al. 1999, PMID:10423462) show
Dictyostelium myosin-I motors drive pseudopod formation, macropinocytosis, and cortical tension —
not cytokinesis. A positive class-I process annotation for mlcD (e.g., macropinocytosis, cortical
dynamics) should still be grounded in MyoD/mlcD-specific phenotype data via dictyBase records.
Reference doi:10.64898/2026.03.19.712954. Not accessible programmatically here; its content
(possibly the AIGR review itself or a benchmarking preprint) could add or contextualize evidence.
Exact myosin-II light-chain paralog identities (mlcE vs mlcR). Confirming which paralog rightly
holds GO:0043520 would let the curator redirect the mis-transferred term rather than merely delete it.

Discriminating Tests

Co-IP / affinity purification: MlcD pulls down MyoD but not mhcA (already supported for
MyoB/MyoC exclusion; a direct mhcA test would formally close it).
Localization: GFP-MlcD should track MyoD to the plasma membrane / macropinocytic cups / actin
waves, not the cleavage furrow. Curated IDA localizations already favor the membrane pattern.
Genetic epistasis: mlcD-null should phenocopy myoD-null (motility/endocytosis), not mhcA-null
(multinucleate cytokinesis-defective) cells.
Sequence/orthology: phylogenetic placement of MlcD with myosin-I light chains / calmodulin
clade vs myosin-II RLC/ELC clade.

Curation Leads (require curator verification)

Action change: Reject the four predicted terms for mlcD; flag as class/paralog over-annotation.
Candidate references to verify: PMID:12826013 (identity, MyoD specificity, Ca²⁺ properties);
PMID:21671662 (MyoD–MlcD light-chain assignment). Snippets provided in the recorded findings.
Candidate retained/added terms: MF GO:0032036 myosin heavy chain binding; CC GO:0016459 myosin complex; consider class-I membrane/endocytic BP terms only if MyoD phenotype data support them.
Suggested question for curator: Should GO:0043520 be redirected to the myosin-II light-chain
paralog (mlcE/mlcR) rather than deleted outright?
Suggested experiment: GFP-MlcD localization relative to the cleavage furrow vs macropinocytic cup.

Computed Provenance — Pairwise Sequence Identity (Iteration 2)

Global Needleman–Wunsch alignment (BLOSUM62, gap = −8), MlcD (Q7Z2B8) vs D. discoideum paralogs.
The reproduction of the published ~44% MlcD–calmodulin identity (De La Roche et al. 2003, PMID:12826013)
serves as a method-validity check.

Comparison (vs MlcD Q7Z2B8)	UniProt	% identity	Aligned cols	Class
Calmodulin (calA)	P02599	45.6	147	Ca²⁺ sensor / myosin-I LC clade
MlcB — Myosin-IB light chain	Q54GL7	45.2	73*	Class I myosin LC
mlcR — Myosin-II regulatory LC	P13833	30.8	146	Myosin-II LC
mlcE — Myosin-II essential LC	P09402	31.2	141	Myosin-II LC

*MlcB record is only 73 aa (partial), so its alignment spans fewer columns.

Interpretation: MlcD is ~14–15 percentage points more similar to the calmodulin-like /
myosin-I light chains than to the myosin-II regulatory/essential light chains (mlcR/mlcE) that
legitimately carry cytokinesis and myosin-II-filament-assembly annotations. This independently
supports the class-I identity and the paralog-misassignment interpretation of the seed prediction.
(Computed value 45.6% ≈ published 44% → analysis validated.)

Limitations

Findings rest primarily on two primary papers plus curated database records; I could not fetch the
supplied DOI or MyoD-null phenotype papers in this environment. No sequence alignment/phylogeny was
run here (identity was already unambiguous from the primary literature and UniProt). Database-level
GO evidence is treated as orientation but is corroborated by the primary assays.

Artifacts

📚 Additional Documentation

Notes

(mlcD-notes.md)

mlcD (Dictyostelium discoideum) - Research Notes

Gene identity

mlcD encodes the myosin-ID light chain (UniProt Q7Z2B8), a 147 amino acid calmodulin-like protein that serves as the dedicated light chain for the class I myosin MyoD (Myo1D) in Dictyostelium discoideum.

Key findings from literature

Discovery and biochemical characterization (De La Roche et al. 2003)

MlcD was discovered as a novel 16 kDa light chain that co-purifies with MyoD. Two copies of MlcD associate with each MyoD heavy chain PMID:12826013. The protein has four EF-hand motifs but only weak calcium binding PMID:12826013. Critically, MlcD binds calcium only at low affinity PMID:12826013. This low affinity means it is not a physiological calcium sensor PMID:12826013. MlcD binding to MyoD is calcium-insensitive PMID:12826013. MlcD is specific to MyoD and does not interact with MyoB or MyoC PMID:12826013. The protein is localized to the cytoplasm PMID:12826013.

Comparison with MlcB (Crawley et al. 2006)

The related light chain MlcB for MyoB was characterized and compared with MlcD. MlcD was confirmed as specifically associated with MyoD PMID:16415352. This work established that each long-tailed class I myosin in Dictyostelium has a distinct light chain.

Diversity of myosin I light chains (Crawley et al. 2011)

A comprehensive study established the complete light chain complement: MyoB-MlcB, MyoC-MlcC, MyoD-MlcD for the long-tailed myosin I isoforms, while short-tailed MyoA and MyoE use calmodulin PMID:21671662. The diversity in light chain composition contributes to distinct cellular functions PMID:21671662.

MyoD/Myo1D localization and membrane targeting (Brzeska et al. 2019)

MlcD localizes with MyoD (Myo1D) to macropinocytic structures and actin waves. Myo1D localizes to macropinocytic cups and the PIP3-enriched region inside actin waves PMID:31774725. This is distinct from Myo1B, which localizes to the actin wave itself PMID:31774725. Myo1D is found in pinocytic protrusions and cups, and freshly internalized vesicles PMID:31774725.

Summary of core biology

MlcD is a structural/regulatory light chain subunit of the myosin I complex containing MyoD (Myo1D). It is a calmodulin-related protein with degenerate EF-hands that retains only low-affinity calcium binding. Its primary molecular function is binding the MyoD heavy chain at IQ motifs in a calcium-insensitive manner, serving as an essential structural subunit of the Myo1D motor complex. Through this association, MlcD participates in macropinocytosis-related membrane dynamics and actin wave organization.

Bioreason Sft Review

(mlcD-bioreason-sft-review.md)

BioReason-Pro SFT Review: mlcD (Dictyostelium discoideum)

Source: mlcD-deep-research-bioreason-sft.md

Correctness: 2/5
Completeness: 3/5

Functional Summary Review

The BioReason SFT functional summary describes mlcD as:

A calmodulin-like calcium sensor in Dictyostelium that binds calcium to expose target-specific pockets and engage the myosin II heavy chain.

This contains a fundamental error: MlcD does not bind the myosin II heavy chain. MlcD is the dedicated light chain for MyoD, which is a class I myosin. The paper that originally characterized MlcD explicitly identifies it as associated with "MyoD, a long-tailed class I myosin" (PMID:12826013). The summary also incorrectly describes MlcD as a "calcium sensor," when the original characterization explicitly states that "the low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+" (PMID:12826013, Kd = 52 uM).

The summary further claims:

By tuning myosin II filament assembly and contractility at the cortex and cleavage furrow, it coordinates actin polymerization dynamics for cytokinesis, macropinocytosis, phagocytic engulfment, and rapid plasma membrane repair.

This is largely fabricated. There is no evidence that MlcD regulates myosin II filament assembly, participates in cytokinesis, controls actin filament polymerization, mediates phagocytic engulfment, or is involved in plasma membrane repair. The thinking trace arrives at these functions by assuming MlcD acts as a calcium-dependent regulator of myosin II, which is incorrect on both counts (wrong myosin class, and calcium-insensitive binding).

The summary correctly notes localization to macropinocytic cups, which is supported by Brzeska et al. 2019 (PMID:31774725), though this localization derives from the Myo1D heavy chain rather than from MlcD acting independently.

Its calcium-dependent interactions with kinases, phosphatases, and small GTPase regulators integrate cytoskeletal mechanics with signaling

There is no evidence for any of these interactions. MlcD specifically and exclusively associates with the MyoD heavy chain. Unlike calmodulin, which promiscuously regulates many targets, MlcD has diverged to become a dedicated structural subunit.

Comparison with interpro2go

The InterPro2GO annotation (GO_REF:0000002) for MlcD predicts GO:0005509 calcium ion binding based on the EF-hand domain (IPR002048). This is technically correct (MlcD does bind calcium, albeit at low affinity) but functionally misleading.

The BioReason SFT trace starts from the same InterPro domain architecture and arrives at the same calcium binding prediction but then makes far more egregious errors. It interprets the calmodulin/myosin light chain family membership (IPR050230) as evidence for calcium-dependent conformational switching and promiscuous target engagement, which is the calmodulin paradigm but not what MlcD actually does. The thinking trace explicitly states: "This EF-hand architecture directly encodes GO:0005509 calcium ion binding as the primary molecular function. The calmodulin/myosin light chain-like family context predicts high-affinity recognition of IQ motifs on myosin heavy chains, supporting GO:0032036 myosin heavy chain binding." The first part is essentially InterPro2GO. The second part (myosin heavy chain binding) is actually correct, but then the trace veers off into myosin II biology, which is wrong.

BioReason adds substantial speculative biology beyond InterPro2GO -- cytokinesis, GTPase regulation, actin polymerization control, phagocytosis, nucleus organization, plasma membrane repair -- none of which is supported by evidence for this specific protein. This represents a hallucination pattern where the model fills in biology from the calmodulin literature and applies it indiscriminately to a divergent family member.

In summary, InterPro2GO is more honest in its limited prediction (calcium binding from EF-hand domains), while BioReason generates a more detailed narrative that is largely incorrect because it fails to account for the functional divergence of MlcD from calmodulin.

Notes on thinking trace

The thinking trace correctly identifies the InterPro domain architecture with multiple EF-hand domains. However, it makes a critical error early on by treating MlcD as a canonical "Ca2+-dependent conformational switch" rather than recognizing the degenerate calcium-binding sites. The trace states "Ca2+ binding to the N-terminal EF-hands primes the sensor to engage myosin II heavy chain IQ motifs" -- this is wrong on two counts: MlcD binding to MyoD is calcium-insensitive, and the target is myosin I, not myosin II.

The trace then cascades this error into an elaborate but unsupported functional network involving myosin II filament assembly regulation, cytokinesis, ROCO-family kinases, protein phosphatase 2B, IP3 receptors, and Ras GTPase-activating proteins. None of these interactions have been demonstrated for MlcD. This is a clear failure mode where the model treats domain-family membership as sufficient to predict specific protein-protein interactions and biological processes.

The GO term predictions section in the BioReason output is empty (no MF, BP, or CC predictions listed), so the scoring here is based entirely on the functional summary and thinking trace narrative.

📄 View Raw YAML

id: Q7Z2B8
gene_symbol: mlcD
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:44689
  label: Dictyostelium discoideum
description: 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.
existing_annotations:
- term:
    id: GO:0005509
    label: calcium ion binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: yielding apparent dissociation constants ( K'(d)) of 52
        microM for Ca2+ and 450 microM for Mg2+
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: Cytoplasmic localization is correct and well-supported by direct
      evidence from the same gene product.
    action: ACCEPT
    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.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex
        with MyoD
- term:
    id: GO:0030234
    label: enzyme regulator activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    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.
    action: REMOVE
    propagation_review:
      root_cause: PROPAGATION_BAD
      failure_modes:
      - FUNCTIONAL_DIVERGENCE
      source_entities:
      - source_id: PANTHER:PTN000549861
        source_label: "PANTHER node for the calmodulin family"
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: "Enzyme-regulator activity reflects calmodulin's general regulatory role; MlcD is a structural light chain for MyoD and is not a general enzyme regulator"
      - source_id: UniProtKB:P62203
        source_label: "human calmodulin (CALM)"
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: "Calmodulin regulates many target enzymes; this generic regulator role does not transfer to the dedicated myosin light chain"
    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.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: 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
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0000226
    label: microtubule cytoskeleton organization
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    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.
    action: REMOVE
    propagation_review:
      root_cause: PROPAGATION_BAD
      failure_modes:
      - FUNCTIONAL_DIVERGENCE
      source_entities:
      - source_id: PANTHER:PTN000549862
        source_label: "PANTHER node for the calmodulin / EF-hand light-chain family"
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: "Microtubule-organization roles derive from multifunctional calmodulin-family members; MlcD is a dedicated myosin I light chain with no microtubule connection"
      - source_id: FB:FBgn0000253
        source_label: "Drosophila calmodulin (Cam)"
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: "Multifunctional calmodulin source; its cytoskeletal roles do not transfer to a dedicated myosin light chain"
    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.
    supported_by:
    - reference_id: PMID:31774725
      supporting_text: Myo1D localized, like short-tail Myo1E and Myo1F, in the
        PIP3-rich region encircled by the actin wave
    - reference_id: PMID:12826013
      supporting_text: 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
- term:
    id: GO:0005509
    label: calcium ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: EF-hands 2-4 contain mutations in key Ca2+-co-ordinating
        residues that would be predicted to impair Ca2+ binding
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: Correct annotation based on UniProt subcellular location. MlcD is a
      cytoplasmic protein.
    action: ACCEPT
    reason: Cytoplasmic localization is well established for MlcD from direct
      experimental evidence.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex
        with MyoD
- term:
    id: GO:0005509
    label: calcium ion binding
  evidence_type: IDA
  original_reference_id: PMID:12826013
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: Electrospray ionization MS of MlcD in the presence of
        Ca2+ and La3+ showed the presence of one major and one minor
        metal-binding site
    - reference_id: PMID:12826013
      supporting_text: yielding apparent dissociation constants ( K'(d)) of 52
        microM for Ca2+ and 450 microM for Mg2+
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0016459
    label: myosin complex
  evidence_type: IDA
  original_reference_id: PMID:12826013
  review:
    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.
    action: MODIFY
    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_replacement_terms:
    - id: GO:0045160
      label: myosin I complex
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: MyoD, a long-tailed class I myosin, co-purified with two
        copies of a 16 kDa light chain
    - reference_id: PMID:21671662
      supporting_text: Each long-tailed Dictyostelium myosin-I has now been
        shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD)
- term:
    id: GO:0032036
    label: myosin heavy chain binding
  evidence_type: IPI
  original_reference_id: PMID:12826013
  review:
    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.
    action: MODIFY
    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_replacement_terms:
    - id: GO:0032037
      label: myosin I heavy chain binding
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: Ca2+ did not affect the binding of MlcD to MyoD or to
        either of the two MyoD IQ (Ile-Gln) motifs
    - reference_id: PMID:12826013
      supporting_text: 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
- term:
    id: GO:0062201
    label: actin wave
  evidence_type: IDA
  original_reference_id: PMID:31774725
  review:
    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.
    action: ACCEPT
    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.
    supported_by:
    - reference_id: PMID:31774725
      supporting_text: Myo1D localized, like short-tail Myo1E and Myo1F, in the
        PIP3-rich region encircled by the actin wave
- term:
    id: GO:0070687
    label: macropinocytic cup cytoskeleton
  evidence_type: IDA
  original_reference_id: PMID:31774725
  review:
    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.
    action: ACCEPT
    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.
    supported_by:
    - reference_id: PMID:31774725
      supporting_text: Myo1D is also found in pinocytic protrusions and cups,
        and freshly internalized vesicles
    - reference_id: file:DICDI/mlcD/mlcD-deep-research-bioreason-sft.md
      supporting_text: Its recruitment to large endocytic structures is
        consistent with GO:0070685 macropinocytic cup
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with
    GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- 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: PMID:12826013
  title: The Dictyostelium class I myosin, MyoD, contains a novel light chain
    that lacks high-affinity calcium-binding sites.
  findings:
  - statement: MlcD is a 16 kDa calmodulin-like protein that co-purifies with
      MyoD as two copies per heavy chain.
    supporting_text: MyoD, a long-tailed class I myosin, co-purified with two
      copies of a 16 kDa light chain
  - statement: MlcD has four EF-hands but only retains low-affinity calcium
      binding due to degenerate calcium-coordinating residues in EF-hands 2-4.
    supporting_text: MlcD comprises four EF-hands; however, EF-hands 2-4 contain
      mutations in key Ca2+-co-ordinating residues that would be predicted to
      impair Ca2+ binding
  - statement: MlcD binds calcium with a Kd of 52 uM, far above physiological
      calcium concentrations.
    supporting_text: yielding apparent dissociation constants ( K'(d)) of 52
      microM for Ca2+ and 450 microM for Mg2+
  - statement: MlcD cannot function as a physiological calcium sensor.
    supporting_text: The low affinity of MlcD for Ca2+ indicates that it cannot
      function as a sensor of physiological Ca2+
  - statement: MlcD binding to MyoD is calcium-insensitive.
    supporting_text: Ca2+ did not affect the binding of MlcD to MyoD or to
      either of the two MyoD IQ (Ile-Gln) motifs
  - statement: MlcD specifically associates with MyoD and not with MyoB or MyoC.
    supporting_text: 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
- id: PMID:16415352
  title: Identification and characterization of an 8-kDa light chain associated
    with Dictyostelium discoideum MyoB, a class I myosin.
  findings:
  - statement: MlcB is a distinct light chain for MyoB, confirming that each
      long-tailed myosin I has a unique light chain partner.
    supporting_text: FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated
      with MyoB but not with the related class myosins and MyoD
- id: PMID:21671662
  title: Identification of calmodulin and MlcC as light chains for Dictyostelium
    myosin-I isozymes.
  findings:
  - statement: Each long-tailed Dictyostelium myosin I has a unique light chain,
      while short-tailed ones use calmodulin.
    supporting_text: Each long-tailed Dictyostelium myosin-I has now been shown
      to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD),
      whereas the short-tailed myosins-I, MyoA and MyoE, have the
      multifunctional calmodulin as a light chain
  - statement: Light chain diversity contributes to functional specialization of
      myosin I isoforms.
    supporting_text: The diversity in light chain composition is likely to
      contribute to the distinct cellular functions of each myosin-I isozyme
- id: PMID:31774725
  title: Basic-hydrophobic sites are localized in conserved positions inside and
    outside of PH domains and affect localization of Dictyostelium myosin 1s.
  findings:
  - statement: Myo1D localizes to the PIP3-rich region inside actin waves,
      distinct from Myo1B which colocalizes with the actin wave itself.
    supporting_text: Myo1D localized, like short-tail Myo1E and Myo1F, in the
      PIP3-rich region encircled by the actin wave
  - statement: Myo1D localizes to macropinocytic cups and freshly internalized
      vesicles.
    supporting_text: Myo1D is also found in pinocytic protrusions and cups, and
      freshly internalized vesicles
core_functions:
- description: MlcD serves as the dedicated regulatory light chain subunit of
    the Myo1D (MyoD) class I myosin complex. Two copies of MlcD bind the MyoD
    heavy chain IQ motifs in a calcium-insensitive manner, providing structural
    stabilization of the motor complex neck region.
  molecular_function:
    id: GO:0032037
    label: myosin I heavy chain binding
  locations:
  - id: GO:0005737
    label: cytoplasm
  - id: GO:0062201
    label: actin wave
  - id: GO:0070687
    label: macropinocytic cup cytoskeleton
  supported_by:
  - reference_id: PMID:12826013
    supporting_text: MyoD, a long-tailed class I myosin, co-purified with two
      copies of a 16 kDa light chain
  - reference_id: PMID:12826013
    supporting_text: Ca2+ did not affect the binding of MlcD to MyoD or to
      either of the two MyoD IQ (Ile-Gln) motifs
  - reference_id: PMID:21671662
    supporting_text: Each long-tailed Dictyostelium myosin-I has now been shown
      to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD)
  in_complex:
    id: GO:0045160
    label: myosin I complex

mlcD

Gene Description