Ryan Vignogna

The yeast DENN domain protein Avl9 contributes to recycling and sorting of endosomal cargos https://www.biorxiv.org/content/10.64898/2026.02.08.704655v1

I owe a very special thanks to Shiying Huang and all members of the Baskin Lab (@jeremybaskin.bsky.social) for helping this yeasty boy learn the ins and outs of cell culture.
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More broadly, I think this work is a nice example of the power of AlphaFold in identifying both previously-unknown protein-protein interactions and protein functions.

Overall, this work identifies a new class of monomeric “DENN GAP” proteins, with Avl9 and DENND6A as its founding members. Future work is aimed at determining the substrates and biological roles of the remaining DENN GAPs.

We tested one of those candidates, DENND6A, and found that it indeed possesses strong GAP activity towards the Arf-like GTPase ARL8B:

Based on these findings, we predicted that other monomeric DENN domain proteins may be Arf-GAPs.
We predicted that other yeast (Anr2 + Afi1) and human (DENND11 + DENND6A/B) proteins are likely Arf-GAPs:

Avl9 GAP function is (likely) conserved in humans.
Mutation of the predicted catalytic arginine in human AVL9 abolishes its ability to promote cell migration, linking its enzymatic activity to cancer physiology:

Additional structural predictions hinted that Avl9 also interacts with a Rab.
We found that Avl9 localization is mediated by Rab8, which binds a distinct, non-catalytic surface of Avl9. Rab8 recruitment strongly enhances Avl9’s Arf1-GAP activity on membranes:

Biochemical assays confirmed this prediction.
Avl9 exhibits robust GAP activity toward Arf-family GTPases, with highest activity toward Arf1:

Using large-scale AlphaFold-multimer–based interaction screens, we identified a high-confidence interaction between Arf1 and Avl9. The predicted interaction resembles that of a GTPase–GAP interaction, with a conserved arginine finger of Avl9 pointed at Arf-bound GTP:

Monomeric DENN domain proteins are widely thought to function solely as Rab-GEFs, yet the molecular function of Avl9 has remained unclear despite its role in secretion and cell migration.

Avl9 defines a family of GTPase-activating proteins that regulate diverse cell biological functions Ras-related GTPases are molecular switches regulating hundreds of signaling and trafficking pathways in cells. Many GTPase regulators remain to be identified despite extensive genetic and biochemical ...

Mind the GAP 🚧🕳️🚧
Excited to share the latest preprint from the Fromme Lab (@fromme-lab.bsky.social), showing that the DENN domain protein Avl9 functions as an Arf-GAP, revising long-standing assumptions about DENN protein function, 🧵:
www.biorxiv.org/content/10.6...

Excited to share the published version of our work by @ryanfeathers.bsky.social and @rvig.bsky.social on the structural basis for activation of Rab6 by the Ric1-Rgp1 complex! rdcu.be/d2mu3

Here is an updated thread about the paper we first shared on that older platform when we posted the preprint