Biochemical interaction of an actin-capping protein, CapZ, with NAP-22.
NAP-22 is a neuronal protein localized in the presynaptic membrane and synaptic vesicles and recovered in a Triton-insoluble low-density microdomain fraction after biochemical fractionation of the synaptic plasma membrane. NAP-22 organizes membrane microdomains through binding to membrane lipids such as cholesterol, phosphatidylethanolamine, and phosphatidylinositol 4,5-bisphosphate. In this study, NAP-22-binding proteins were screened through the pull-down assay using brain-derived NAP-22 bound to Sepharose 4B. An actin-capping protein, CapZ, was identified in the precipitate through mass spectrometry and Western blotting. CapZ was then expressed in E. coli and the purified protein-bound NAP-22 directly. Because bacterially expressed NAP-22 bound CapZ, it was determined that the N-terminal myristoyl moiety of NAP-22 is not necessary for the binding. The binding of NAP-22 showed no effect on the actin nucleation activity of CapZ measured with centrifugation and viscometric assays. Hence, the CapZ-NAP-22 complex could work as the nucleation site of actin polymerization or as the actin filament-anchoring site on the membrane microdomain.
Odagaki
Sin-Ichi
S
Division of Biology, Graduate School of Science, Kobe University, Nada-ku, Kobe, Japan.
Kumanogoh
Haruko
H
Nakamura
Shun
S
Maekawa
Shohei
S
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000096:biosynthesis; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D001923:Brain Chemistry / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D051347:CapZ Actin Capping Protein / Q000737:chemistry* / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D002148:Calmodulin-Binding Proteins; D051347:CapZ Actin Capping Protein; D003598:Cytoskeletal Proteins; D008563:Membrane Lipids; D009419:Nerve Tissue Proteins; C066853:Basp1 protein, rat