Lipid binding activity of a neuron-specific protein NAP-22 studied in vivo and in vitro.
There exists a microdomain called "raft" in the cell membrane. The enrichment of cholesterol and sphingolipids in its outer leaflet is well recognized. In contrast, little is known of the lipid composition of the inner leaflet of raft, where many acylated signal-transducing molecules, such as trimeric G proteins and protein tyrosine kinases, associate. NAP-22 is a neuronal protein localized on the inner leaflet of raft domain. This protein was found to bind cholesterol in the liposome. In this study, we further analyze the lipid binding activity of NAP-22 using eukaryotic and bacterial expression systems. In addition to cholesterol, NAP-22 showed a phosphatidylethanolamine (PE)- and polyphosphoinositide-dependent membrane binding in the liposome assay. The N-terminal myristoylation was essential for the liposome binding. The C-terminal deletion up to D61 showed little effect on the binding. The lipid binding region was hence judged to be in the N-terminal 60-amino-acid sequence. NAP-22 was then expressed in COS7 cells, and the intracellular localization was studied. Biochemical analysis showed the localization of NAP-22 in a Triton-insoluble low-density fraction. Cell staining analysis showed colocalization patterns of NAP-22 with PE and cholesterol in the membrane.
Terashita
Akira
A
Division of Bioinformation, Department of Life Science, Graduate School of Science and Technology, Kobe-University, Rokkodai, Nada-ku, Kobe, Japan.
Funatsu
Nobuo
N
Umeda
Masato
M
Shimada
Yukiko
Y
Ohno-Iwashita
Yoshiko
Y
Epand
Richard M
RM
Maekawa
Shohei
S
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003598:Cytoskeletal Proteins; D007150:Immunohistochemistry; D050356:Lipid Metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000737:chemistry
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D008081:Liposomes; D009419:Nerve Tissue Proteins; D010714:Phosphatidylethanolamines; C066853:Basp1 protein, rat; D002784:Cholesterol