WDR41

WDR41 is a β-propeller scaffold protein that functions as an essential subunit of the C9orf72–SMCR8–WDR41 (CSW) heterotrimeric complex, coupling lysosomal amino acid sensing to autophagy initiation, mTORC1 signaling, and GTPase regulation. WDR41 binds the DENN domain of SMCR8 via its N-terminal β-strand and C-terminal helix—without directly contacting C9orf72—and recruits the entire complex to lysosomes by engaging the cationic amino acid transporter PQLC2 through a short flexible loop motif that inserts into a cavity exposed only in the inward-facing, substrate-free conformation of PQLC2, thereby transducing lysosomal arginine/lysine/histidine availability into complex localization (PMID:32848248, PMID:31851326, PMID:33597295). Once at the lysosome, the C9orf72–SMCR8 longin-domain heterodimer within the complex provides GAP activity preferentially toward ARF1 (and to a lesser extent RAB8A/RAB11A), with SMCR8 Arg147 serving as the catalytic arginine finger, while the complex also associates with the ULK1–FIP200 autophagy initiation machinery to regulate autophagic flux (PMID:34145292, PMID:32303654, PMID:27617292). Loss of WDR41 phenocopies SMCR8 deficiency, causing accumulation of LysoTracker-positive vesicles, delayed phagosome maturation, excessive endosomal TLR signaling, impaired mTORC1 reactivation, and increased lysosomal exocytosis (PMID:30442666, PMID:29995611, PMID:29950492).

1. 2016 High

   Identification of WDR41 as a stable subunit of a trimeric complex with C9orf72 and SMCR8 established the CSW complex as a functional unit linked to RAB GEF activity and autophagy regulation, answering the question of whether C9orf72 acted alone or as part of a defined multiprotein assembly.

   Evidence Reciprocal co-immunoprecipitation, in vitro GEF assays for RAB8a/RAB39b, and knockdown/KO studies with autophagic flux readouts across multiple independent laboratories

   PMID:27103069 PMID:27193190 PMID:27494456 PMID:27617292

   Open questions at the time

   • GEF versus GAP activity assignment was later revised; the initial GEF claim for RABs was not independently confirmed with purified components
   • Direct binding topology among the three subunits was unknown
   • Identity of the lysosomal recruitment mechanism was unresolved
2. 2016 High

   Demonstrating that the CSW complex associates with the ULK1–FIP200 autophagy initiation complex and regulates ULK1 expression placed the complex at the earliest step of autophagosome formation, answering how it connects to the core autophagy machinery.

   Evidence Co-immunoprecipitation with ULK1/ATG101, Smcr8 knockout mouse with autophagy defects

   PMID:27617292

   Open questions at the time

   • Whether WDR41 itself directly contacts ULK1/FIP200 or is required only for complex integrity was not resolved
   • Stoichiometry of the CSW–ULK1 supercomplex was not determined
3. 2018 Medium

   Showing that WDR41 knockout phenocopies SMCR8 loss—with lysosomal vesicle accumulation, delayed phagosome maturation, and excessive TLR signaling—established that WDR41 is functionally non-redundant and essential for the complex's role in endolysosomal trafficking and innate immune regulation.

   Evidence WDR41 KO cells with LysoTracker staining, phagosome maturation kinetics, and inflammatory cytokine assays

   PMID:30442666

   Open questions at the time

   • Whether the phenotypes reflect loss of WDR41-specific function or destabilization of the entire complex was not distinguished
   • Downstream GTPase targets responsible for these phenotypes were not identified
4. 2018 High

   Demonstrating that WDR41 is required for amino-acid-starvation-induced recruitment of the CSW complex to lysosomes and for mTORC1 reactivation—rescued by constitutive lysosomal targeting of C9orf72—answered the question of WDR41's non-enzymatic role: it is the lysosomal targeting subunit.

   Evidence WDR41 KO, lysosome fractionation, mTORC1 activity assays, epistatic rescue with lysosome-targeted C9orf72

   PMID:29995611

   Open questions at the time

   • The lysosomal receptor for WDR41 was unknown
   • Structural basis for WDR41's targeting function was unresolved
5. 2020 High

   Identification of the lysosomal transporter PQLC2 as the WDR41 receptor, and demonstration that arginine/lysine/histidine negatively regulate this interaction, revealed the amino acid sensing logic: cationic amino acid depletion from lysosomes exposes the WDR41 binding site on PQLC2.

   Evidence Reciprocal co-immunoprecipitation, lysosome fractionation, amino acid titration of WDR41–PQLC2 binding

   PMID:31851326

   Open questions at the time

   • The structural details of the WDR41–PQLC2 interface were not yet resolved
   • Whether PQLC2 conformational state directly controls WDR41 access was not proven
6. 2020 High

   Cryo-EM structures of the CSW complex resolved WDR41 as a β-propeller contacting only the SMCR8 DENN domain, corrected the enzymatic assignment from GEF to GAP (toward ARF GTPases), and showed the complex forms a dimer of heterotrimers with SMCR8 Arg147 as the catalytic arginine finger.

   Evidence Cryo-EM at 3.2–3.8 Å resolution, in vitro GAP assays, interface and active-site mutagenesis

   PMID:32303654 PMID:32848248

   Open questions at the time

   • Physiological ARF substrates in vivo were not validated
   • The biological function of complex dimerization was unclear
   • WDR41's contribution to catalysis beyond scaffolding was not defined
7. 2021 High

   Fine mapping of the WDR41–PQLC2 interface showed that a short peptide motif in a flexible WDR41 loop inserts into a cavity exposed only in the inward-facing conformation of PQLC2, establishing a transceptor mechanism whereby transporter conformational cycling gates signaling complex recruitment.

   Evidence Structure-guided mutagenesis of the WDR41 loop motif, co-immunoprecipitation

   PMID:33597295

   Open questions at the time

   • No high-resolution structure of the WDR41–PQLC2 interface exists
   • Whether other transporters can substitute for PQLC2 in other tissues is untested
8. 2021 High

   A substrate-bound cryo-EM structure of ARF1-GDP-BeF₃⁻ with the CSW complex showed the SMCR8/C9orf72 longin domains form the ARF1 binding pocket and confirmed ARF1 as the preferred GAP substrate (~10-fold lower Km than RAB8A), resolving the long-standing question of enzymatic specificity.

   Evidence Cryo-EM of the transition-state complex, quantitative in vitro GAP kinetics, active-site and interfacial mutagenesis

   PMID:34145292

   Open questions at the time

   • In vivo ARF1 regulation by the CSW complex has not been validated in cellular systems
   • Roles of specific ARF substrates in autophagy or lysosomal function downstream of the complex remain uncharacterized

• Key open questions include: (1) how CSW complex GAP activity toward ARF GTPases mechanistically connects to its roles in autophagy initiation, mTORC1 signaling, and lysosomal exocytosis; (2) whether WDR41 has functions independent of the CSW complex; and (3) the structural basis of the WDR41–PQLC2 interface at atomic resolution.
• No high-resolution structure of the WDR41–PQLC2 binary complex
• ARF-dependent downstream effectors mediating CSW phenotypes are unidentified
• WDR41-independent functions remain unexplored