Amino acid sequence motifs and mechanistic features of the membrane translocation of alpha-synuclein.
Many lines of evidence suggest that alpha-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of alpha-synuclein, and the mechanistic features of the phenomenon. We first showed that not only alpha-synuclein but also beta- and gamma-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of alpha-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. alpha-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of alpha-synuclein at 4 degrees C was comparable with the import observed at 37 degrees C. Furthermore, membrane translocation of alpha-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of alpha-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.
Ahn
Keun Jae
KJ
Department of Microbiology and Brain Korea 21 Projects For Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Paik
Seung R
SR
Chung
Kwang Chul
KC
Kim
Jongsun
J
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000502:physiology; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D013696:Temperature; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism*; D051846:beta-Synuclein / Q000378:metabolism; D051845:gamma-Synuclein / Q000378:metabolism
D008565:Membrane Proteins; D010455:Peptides; D051844:alpha-Synuclein; D051846:beta-Synuclein; D051845:gamma-Synuclein