The intermolecular interaction between the PH domain and the C-terminal domain of Arabidopsis dynamin-like 6 determines lipid binding specificity.
Dynamin and its related proteins are a group of mechanochemical proteins involved in the modulation of lipid membranes in various biological processes. Here we investigate the nature of membrane binding of the Arabidopsis dynamin-like 6 (ADL6) involved in vesicle trafficking from the trans-Golgi network to the central vacuole. Fractionation experiments by continuous sucrose gradients and gel filtration revealed that the majority of ADL6 is associated with membranes in vivo. Amino acid sequence analysis revealed that ADL6 has a putative pleckstrin homology (PH) domain. In vitro lipid binding assays demonstrated that ADL6 showed high affinity binding to phosphatidylinositol 3-phosphate (PtdIns-3-P) and that the PH domain was responsible for this interaction. However, the PH domain alone binds equally well to both PtdIns-3-P and phosphatidylinositol 4-phosphate (PtdIns-4-P). Interestingly, the high affinity binding of the PH domain to PtdIns-3-P was restored by a protein-protein interaction between the PH domain and the C-terminal region. In addition, deletion of the inserted regions within the PH domain results in high affinity binding of the PH domain to PtdIns-3-P. These results suggest that ADL6 binds specifically to PtdIns-3-P and that the lipid binding specificity is determined by the interaction between the PH domain and the C-terminal domain of ADL6.
Lee
Sung Hoon
SH
Center for Plant Intracellular Trafficking and the Division of Molecular and Life Sciences, Pohang University of Science and Technology, 790-784, Korea.
Jin
Jing Bo
JB
Song
Jinhee
J
Min
Myung Ki
MK
Park
Dae Sup
DS
Kim
Yong-Woo
YW
Hwang
Inhwan
I
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D017931:DNA Primers; D007295:Inositol Phosphates; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C434467:ADL6 protein, Arabidopsis; D020558:GTP Phosphohydrolases; D034281:Dynamins