RasC and Macropinocytosis (GO:0044351) in *Dictyostelium discoideum* — Focused Curation Report OpenScientist openscientist-autonomous 2 artifacts 2026-09-20T20:15:12.548908

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RasC and Macropinocytosis (GO:0044351) in Dictyostelium discoideum — Focused Curation Report

Gene: rasC (DICDI) · UniProt: P32253 · Hypothesis slug: function-hypothesis-go-0044351
Focus type: function_assignment
Seed hypothesis: Dictyostelium discoideum RasC participates in macropinocytosis (GO:0044351). Distinguish positive/negative regulatory contributions, direct machinery roles, compensatory Ras paralogs, and secondary growth effects. An increase in fluid uptake after deletion does not by itself establish lack of a role.

Executive Judgment

Verdict: Weakly supported / over-annotated for a direct role. There is no primary experimental evidence that RasC directly participates in macropinocytosis. Across the entire primary macropinocytosis literature in Dictyostelium, the Ras proteins repeatedly and specifically implicated are RasG, RasS, and RasB — never RasC. RasC's experimentally validated function is a distinct axis: RasGEFA(Aimless)→RasC→TORC2→PKB, driving cAMP relay (adenylyl cyclase activation) and chemotaxis. Direct paralog-substitution data show RasC cannot replace RasG in growth in suspension, a macropinocytosis-dependent process.

The current GO:0044351 annotation on P32253 carries the evidence code IBA (Inferred from Biological Ancestor, GO_Central) — a phylogenetic projection, not experimental support. Given that RasC is ~66% identical to RasG (the validated macropinocytosis regulator) and sits in the same catalytic Ras clade, the annotation is best explained as clade-level paralog carry-over.

Most important caveat (honoring the seed): Absence of a published rasC⁻ macropinocytosis assay means a subtle or negative-regulatory contribution cannot be formally excluded. The seed's warning — that increased fluid uptake after deletion would not by itself disprove a role — is methodologically valid, but here there is no such deletion-uptake dataset in evidence either way; the case against a direct machinery role rests on positive identification of other paralogs plus a direct negative substitution result. This warrants not asserting a direct RasC macropinocytosis function while flagging the IBA term as a candidate for removal/generalization pending curator review.

Evidence Matrix

Citation (PMID)	Evidence type	Direction	Claim tested	Key finding	Context	Confidence / limitations
30967009 (Williams, Paschke, Kay 2019)	Review of genetics + gain/loss-of-function	Competing / refutes direct RasC role	Which Ras drive macropinocytosis	"RasG and RasS are the key Ras proteins involved"; GefF implicated GEF	D. discoideum, growing cells	High; review-level synthesis of primary data, RasC not mentioned
23843627 (Hoeller et al. 2013)	Direct assay: Ras–PI3K binding + single mutants	Competing / refutes	Which Ras act via PI3K in macropinocytosis	RasG and RasS bind PI3K1/2 & PI3K4; single rasG or rasS mutants have severe macropinocytosis defects	D. discoideum, vegetative	High; direct biochemistry + phenotype; does not test rasC
27821733 (Junemann et al. 2016)	Direct assay: effector regulation	Competing / refutes	Upstream Ras of macropinocytosis formin ForG	ForG directly regulated by RasB and RasG	D. discoideum, macroendocytosis	High; direct; RasC not implicated
38263885 (Putar et al. 2024)	Mutant + localization	Competing / refutes	Major macropinocytosis Ras	"RasG is a major regulator of macropinocytosis"; IqgC loading depends on RasG	D. discoideum	High
20833893 (Bolourani et al. 2010)	Mutant paralog-substitution	Refutes (negative)	Can RasC substitute for RasG in growth in suspension (macropinocytic feeding)?	RasD fully substitutes; RasC is without effect	D. discoideum vegetative	High; direct negative evidence relevant to macropinocytosis-dependent growth
17380187 (Kae et al. 2007)	Direct GEF specificity (in vitro + in vivo)	Qualifies (assigns RasC elsewhere)	RasC pathway specificity	RasGEFA activates RasC for adenylyl cyclase activation; RasG for chemotaxis	D. discoideum development	High; defines RasC's true niche
20493808 / 20660630 / 27172998 (Charest, Cai, Khanna)	Direct assay / interaction	Qualifies	RasC effector	RasC is the upstream activator of TORC2→PKB for chemotaxis/cAMP relay	D. discoideum	High; RasC = TORC2 axis, not macropinocytosis
25815683 (Bloomfield et al. 2015)	Forward genetics / mutant	Context	What limits fluid uptake	NF1 RasGAP loss increases fluid uptake via greater Ras/PI3K activity	D. discoideum axenic strains	High; shows Ras-tone control of uptake but does not implicate RasC specifically
UniProt P32253 (dictyBase/GO_Central)	Database annotation	Qualifies	Current annotation basis	GO:0044351 = IBA; all experimental codes (IDA/IMP/IGI) are TORC2/cAMP/chemotaxis	Annotation record	High; documents phylogenetic, non-experimental basis
24742374 (Chattwood et al. 2014)	Mutant / growth context	Qualifies (supports no essential RasC role)	Does feeding require RasC?	rasG⁻C⁻ double-null cells are routinely grown in axenic medium (which depends on macropinocytic feeding), i.e. bulk fluid uptake persists without RasC	D. discoideum	Medium; incidental to the study's chemotaxis focus, not a quantitative uptake assay
This report (computed)	Computational (sequence)	Structural/evolutionary	Paralog relatedness	RasC↔RasG 65.6%, ↔RasD 64.6%, ↔RasB 58.4%, ↔RasS 53.1% identity (global NW)	In-silico	Medium; simple global alignment, supports paralog-projection interpretation

GO Curation Implications

Lead (requires curator verification):

Term: GO:0044351 macropinocytosis (BP), currently on P32253 via IBA.
Recommended action: Do not retain as an experimentally-styled assertion of RasC function. Treat as non-core / candidate for removal or generalization. The experimental record supports RasG/RasS/RasB for this process, not RasC.
Option A (preferred): Remove the IBA macropinocytosis annotation from RasC, or down-rank it, noting paralog carry-over from RasG.
Option B (conservative): Retain but flag as phylogenetic-only (IBA) with an explicit note that no RasC-specific experimental evidence exists; do not promote to an experimental code.
Terms that ARE experimentally supported and should be emphasized as core: GO:1904841 TORC2 complex binding (IDA), GO:1904515 positive regulation of TORC2 signaling (IDA), GO:0010856 adenylate cyclase activator activity (IGI), GO:0030250 guanylate cyclase activator activity (IMP), GO:0043327 chemotaxis to cAMP (IMP), GO:0007188 adenylate cyclase-modulating GPCR signaling (IMP). These represent RasC's primary function.

"Protein binding" is not used as a recommendation; the informative core MF/BP terms above are supported.

GO Decision Table (lead — requires curator verification)

GO ID	Term (aspect)	Current code on P32253	Evidence for RasC specifically	Recommended curation action
GO:0044351	macropinocytosis (BP)	IBA (phylogenetic)	None experimental; paralog carry-over from RasG	Remove or flag as phylogenetic-only / non-core; do not promote to experimental code
GO:1904841	TORC2 complex binding (MF)	IDA	Direct (RasC–TOR/TORC2 binding)	Retain — core
GO:1904515	positive regulation of TORC2 signaling (BP)	IDA	Direct	Retain — core
GO:0010856	adenylate cyclase activator activity (MF)	IGI	Genetic, RasC-specific	Retain — core
GO:0030250	guanylate cyclase activator activity (MF)	IMP	Mutant phenotype	Retain
GO:0043327	chemotaxis to cAMP (BP)	IMP	Mutant phenotype	Retain — core
GO:0007188	adenylate cyclase-modulating GPCR signaling (BP)	IMP	Mutant phenotype	Retain — core
GO:0031152	aggregation involved in sorocarp development (BP)	IMP	Mutant phenotype	Retain

Mechanistic Scope

Immediate molecular function of RasC: a GTP-binding/GTPase small G-protein that, when activated by its cognate GEF RasGEFA (Aimless), binds and activates TORC2, driving PKB/PKBR1 phosphorylation and adenylyl cyclase A activation (cAMP relay) and chemotaxis to cAMP.
Macropinocytosis machinery (separate module): organized around a plasma-membrane patch of PIP3 + active RasG/RasS/RasB and Rac, recruiting SCAR/WAVE→Arp2/3 and the formin ForG; regulated by GEFs (GefF, RasGEFX) and GAPs (NF1, RGBARG, IqgC, Leep2). RasC is not a documented member of this module.
Downstream/secondary effects to keep separate from a direct role: any effect of rasC deletion on growth or fluid uptake would be a secondary consequence (e.g., altered global Ras tone, compensatory RasD/RasG expression), not evidence of RasC acting in the macropinocytic cup.

Conflicts and Alternatives

Paralog confusion / clade projection (most likely): RasC ~66% identical to RasG. IBA rules project ancestral function across the clade, sweeping RasC into "macropinocytosis" without species/paralog-specific data.
Shared reporter ambiguity: Excitable-network and cup-imaging studies often use RBD (Ras-binding-domain) biosensors that detect active Ras collectively and cannot distinguish RasC from RasG/RasD; such images should not be read as RasC-specific localization to macropinocytic cups.
Compensation masking: Because RasG (and RasD when de-repressed) can cover growth/cytokinesis, a rasC single deletion could appear neutral or even show increased uptake through compensatory paralog activity — the seed's caveat. This is an argument for caution, not for asserting a direct role.
Negative substitution result: Bolourani 2010 is the cleanest conflict with the hypothesis — RasC explicitly fails to substitute for RasG in growth in suspension (macropinocytic feeding), whereas RasD succeeds.

Knowledge Gaps

Gap	What was checked	Why it matters	Resolving evidence
No published rasC⁻ (or rasC-GTP-locked) macropinocytosis / fluid-phase uptake assay	Multiple PubMed query variants (RasC + macropinocytosis/pinocytosis/endocytosis/fluid-phase/growth/axenic) returned no direct RasC uptake assay; no study reports RasC-GTP binding the macropinocytosis effectors PI3K1/2/4 or ForG	Cannot formally exclude a subtle positive or negative-regulatory RasC contribution; also means the seed's "increased fluid uptake after deletion" scenario has no supporting dataset in evidence	TRITC-dextran fluid-uptake time course in rasC⁻, rasC⁻rasG⁻, and RasC(Q62L) vs WT
Whether RasC localizes to macropinocytic cups	UniProt CC terms list plasma membrane/leading edge/vesicle but not cup-specific; imaging uses pan-Ras RBD probes	Localization to cups would be needed for a direct machinery role	RasC-specific (not RBD) live reporter co-imaged with PIP3/PHcrac and SCAR/WAVE
Whether RasC-GTP binds the macropinocytosis PI3Ks (PI3K1/2, PI3K4)	Hoeller 2013 tested RasG/RasS binding, not RasC	Direct effector binding would upgrade from IBA	In vitro Ras–PI3K RBD pulldowns including RasC
Basis of the IBA call	Confirmed evidence code = IBA on P32253	Determines whether curator should trust the annotation	Inspect the GO_Central PAINT tree / ancestral node for the projection

Discriminating Tests

Quantitative fluid-phase uptake (TRITC-dextran/FITC-dextran) and macropinosome sizing in WT vs rasC⁻ vs rasG⁻ vs rasC⁻rasG⁻, plus constitutively active RasC(Q62L). A direct positive role predicts reduced uptake in rasC⁻ and/or increased uptake with RasC(Q62L); a purely compensatory/secondary effect predicts little change (or increase) in rasC⁻ that is explained by paralog upregulation.
RasC-specific live-cell localization (not RBD) relative to PIP3 and SCAR/WAVE cup markers.
Direct effector-binding assays of RasC-GTP with PI3K1/2, PI3K4, and ForG, benchmarked against RasG/RasS/RasB.
Compensation controls: measure RasG/RasD/RasS activation and protein levels in rasC⁻ to interpret any uptake phenotype.
Inspect the GO_Central PAINT annotation tree to see whether the macropinocytosis node was experimentally anchored on RasG and projected to RasC.

Curation Leads (require curator verification)

Candidate action change: Flag/remove or mark-as-phylogenetic the RasC → GO:0044351 (macropinocytosis) IBA annotation; do not treat RasC macropinocytosis as core function.
Candidate references to cite in the review:
PMID 30967009 — snippet to verify: "genetic analysis suggests that RasG and RasS are the key Ras proteins involved."
PMID 23843627 — "RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects."
PMID 27821733 — "ForG is directly regulated in large-scale endocytosis by RasB and RasG."
PMID 38263885 — "RasG is a major regulator of macropinocytosis."
PMID 20833893 — "RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect."
PMID 17380187 — "RasG regulates chemotaxis and RasC is responsible for adenylyl cyclase activation."
Suggested curator questions: (a) Is the GO:0044351 IBA on RasC anchored on an experimentally-annotated RasG node? (b) Is there any unpublished dictyBase phenotype linking rasC⁻ to fluid uptake? (c) Should RasC's TORC2/cAMP annotations be emphasized as the core function set?
Suggested experiments: the discriminating tests above (fluid-uptake panel, RasC-specific imaging, RasC–PI3K binding).

Provenance / Artifacts

Computed Ras paralog global %identity matrix (Needleman-Wunsch): RasC↔RasG 65.6, ↔RasD 64.6, ↔RasB 58.4, ↔RasS 53.1; RasG↔RasD 82.5 (executed in this run).
UniProt P32253 GO cross-reference dump confirming GO:0044351 = IBA and experimental codes limited to TORC2/cAMP/chemotaxis (executed in this run).
Recorded knowledge-state findings #1–#3.

Computational results reported conservatively; simple global alignment used, no structural modeling performed. No fabricated assay results.