Figure 5. Mipp1 is GPI anchored and dephosphorylates extracellular pools of IPs.

(A) Cartoon of Mipp1 protein showing histidine phosphatase (HP) domain and hydrophobic N-and C-termini.

(B) Candidate topologies for Mipp1, which is anchored in the plasma membrane by either its C-terminal hydrophobic span or GPI anchor.

(C-D) Glycosidase treatment of Mipp1 from (C) embryo extracts and from (D) S2 cell extracts.

(E) Extracellular Mipp1 can be detected in unpermeabilized S2 cells expressing Mipp1.

(F) PI-PLC cleavage releases GPI anchored Mipp1 into the extracellular media. P = pellet (cells); S = supernatant (extracellular media)

(G-H) Trachea overexpressing (G) WT mipp1 in a mipp1^KO (btl>mipp1; mipp1^KO) and (H) enzyme dead mipp1 in a mipp1^KO (btl>mipp1^H67A; mipp1^KO) co-istained with Mipp1 (red) and aPKC (apical marker; green).

(I) Trachea overexpressing mipp1-GFP in a mipp1^KO (btl>mipp1-GFP; mipp1^KO) co-stained with (I) Mipp1 and GFP and with (J) GFP and KDEL (ER marker).

(J) Trachea overexpressing mipp1-GFP (btl>mipp1-GFP) co-stained with GFP and KDEL (ER marker).

(K) Toludine-blue stained PAGE gel of IP₆, IP₅, IP₄, IP₃, and polyphosphate (PolyP) as size indicator.

(L) The reaction products of IP₆ added to the media of S2 cells expressing vector, Mipp1, enzyme-dead Mipp1^H67A , or Mipp1-CFP for 30 mins.

See also Figure S4.