ATP9B

ATP9B is a P4-ATPase lipid flippase that localizes exclusively to the trans-Golgi network (TGN) via a Golgi-targeting signal in its N-terminal cytoplasmic region, and uniquely among P4-ATPases, exits the ER independently of CDC50 auxiliary subunits (PMID:21914794). ATP9B forms homomeric and heteromeric complexes with its paralog ATP9A, and heteromeric interaction contributes to Golgi retention of ATP9A (PMID:40234049). The flippase activity of both ATP9B and ATP9A is required for transport of cargo (VSVG) from the Golgi to the plasma membrane in the exocytic pathway, establishing ATP9B as a functional component of Golgi-to-cell-surface trafficking (PMID:40234049).

1. 2011 High

   Establishing where ATP9B acts and how it reaches its destination resolved a key question about P4-ATPase diversity: ATP9B localizes exclusively to the TGN and, unlike most P4-ATPases, exits the ER without requiring CDC50 proteins, with a Golgi-targeting signal mapped to its N-terminal cytoplasmic region.

   Evidence RNAi depletion of CDC50 proteins, chimeric protein constructs, and fluorescence microscopy in mammalian cells

   PMID:21914794

   Open questions at the time

   • The specific lipid substrates flipped by ATP9B at the TGN were not identified
   • The mechanism by which the N-terminal region directs Golgi targeting was not resolved at the sequence or structural level
   • Functional consequences of ATP9B TGN localization for membrane traffic were unknown
2. 2025 High

   Determining the functional role of ATP9B in membrane trafficking revealed that ATP9B and ATP9A form homomeric and heteromeric complexes, that heteromeric complex formation retains ATP9A in the Golgi, and that flippase activity of both proteins is required for VSVG cargo transport from the Golgi to the plasma membrane.

   Evidence Co-immunoprecipitation demonstrating reciprocal complex formation, VSVG transport assays, and flippase-dead mutant analysis in mammalian cells

   PMID:40234049

   Open questions at the time

   • The specific lipid substrates translocated by ATP9B have not been directly identified
   • Whether ATP9B flippase activity has functions beyond exocytic VSVG transport (e.g., in endosomal or other trafficking pathways) is unresolved
   • Structural basis for ATP9A-ATP9B heteromeric interaction is unknown
3. 2025 Medium

   Cryo-EM structures of the close paralog ATP9A revealed a novel outward gating mechanism (driven by TM6-10 movement) distinct from canonical P-type ATPases, with a large phospholipid-binding cavity accommodating bulky headgroups and ATPase activity stimulated by negatively charged phospholipids; these features likely extend to ATP9B given shared CDC50-independence.

   Evidence Cryo-EM at 2.2 Å resolution of ATP9A, ATPase activity assays with lipid substrates, molecular dynamics simulations

   PMID:40876594

   Open questions at the time

   • Structural data are for ATP9A, not ATP9B directly; applicability to ATP9B is inferred from paralog similarity
   • Whether ATP9B shares the same lipid substrate preferences (PS, phosphoinositides) as ATP9A has not been tested
   • No direct cryo-EM structure of ATP9B exists

• Key unresolved questions include the identity of the endogenous lipid substrates translocated by ATP9B, the structural basis of ATP9A-ATP9B heterocomplex formation, and whether ATP9B has roles in trafficking pathways beyond Golgi-to-plasma-membrane exocytosis.
• No direct identification of ATP9B lipid substrates in a reconstituted or cellular system
• No structure of ATP9B or the ATP9A-ATP9B complex
• Physiological consequences of ATP9B loss in animal models are uncharacterized