Phosphorylation mutants elucidate the mechanism of annexin IV-mediated membrane aggregation.
Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probe the structural basis of annexin IV-induced membrane aggregation and the inhibition of this property by protein kinase C phosphorylation. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr6Ala, T6A) phosphorylation of threonine 6 were produced for these studies and compared with wild-type annexin IV. In vitro assays showed that unmodified wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild-type or the T6D mutant, promote vesicle aggregation. Electron crystallographic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopic images of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated a separation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In this orientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergo face-to-face self-association with the annexin layer of a second vesicle. The 2.0-A resolution crystal structure of the T6D mutant showed that the mutation causes release of the N-terminal tail from the protein core. This change would preclude the face-to-face annexin self-association required to aggregate vesicles. The data suggest that reversible complex formation through phosphorylation and dephosphorylation could occur in vivo and play a role in the regulation of vesicle trafficking following changes in physiological states.
Kaetzel
M A
MA
Departments of Molecular and Cellular Physiology and of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Ohio 45220, USA.
Mo
Y D
YD
Mealy
T R
TR
Campos
B
B
Bergsma-Schutter
W
W
Brisson
A
A
Dedman
J R
JR
Seaton
B A
BA
D000409:Alanine / Q000235:genetics; D000818:Animals; D017314:Annexin A4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D020285:Cryoelectron Microscopy; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D013912:Threonine / Q000235:genetics
D017314:Annexin A4; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D013912:Threonine; C042511:1,2-dioleoylphosphatidylserine; D011493:Protein Kinase C; C017251:1,2-oleoylphosphatidylcholine; D000409:Alanine