Phosphorylation Mutants Elucidate the Mechanism of Annexin IV-Mediated Membrane Aggregation

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• 作者：M. A. Kaetzel ; Y. D. Mo ; T. R. Mealy ; B. Campos ; W. Bergsma-Schutter ; A. Brisson ; J. R. Dedman ; and B. A. Seaton
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 3, 2001
• 年：2001
• 卷：40
• 期：13
• 页码：4192 - 4199
• 全文大小：293K
• 年卷期：v.40,no.13(April 3, 2001)
• ISSN：1520-4995

文摘

Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probethe structural basis of annexin IV-induced membrane aggregation and the inhibition of this property byprotein 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, butnot 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 IVproteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopicimages of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated aseparation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In thisorientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergoface-to-face self-association with the annexin layer of a second vesicle. The 2.0-Å resolution crystal structureof 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. Thedata suggest that reversible complex formation through phosphorylation and dephosphorylation couldoccur in vivo and play a role in the regulation of vesicle trafficking following changes in physiologicalstates.