Protein-protein interactions of alpha-synuclein in brain homogenates and transfected cells.
alpha-Synuclein is a highly conserved presynaptic protein with probable roles in normal synaptic development and plasticity as well as neurodegenerative disease, although its molecular function is not yet clear. To identify potential protein binding partners of alpha-synuclein, we performed co-immunoprecipitations using a monoclonal antibody (H3C) against its C-terminus. More than 20 detectable proteins were specifically co-immunoprecipitated from zebra finch and mouse forebrain extracts. One of these, with relative mobility of 55 kDa, was identified through microsequencing as a mixture of alpha- and beta-tubulin. Tubulin was specifically recovered from a mouse forebrain cytosolic extract by a GST/alpha-synuclein fusion protein immobilized on glutathione-Sepharose beads. In the converse experiment, alpha-synuclein bound to a column prepared from purified bovine brain tubulin immobilized upon CNBr-Sepharose. alpha-Synuclein does not appear to bind assembled microtubules, however, as alpha-synuclein did not pellet with polymerized microtubules in a standard assay for microtubule-associated proteins. Likewise, when a fusion construct of alpha-synuclein and green fluorescent protein (GFP) was expressed in African green monkey kidney epithelial (CV-1) cells, the fusion protein did not colocalize with endogenous microtubules. We conclude that alpha-synuclein may interact specifically with heterodimeric tubulin, but not microtubules, in the neuronal cytosol.
Payton
J E
JE
B107 Chemical and Life Science Laboratory, Department of Cell and Structural Biology, University of Illinois, Urbana, IL, USA.
Perrin
R J
RJ
Clayton
D F
DF
George
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D016548:Prosencephalon / Q000378:metabolism*; D011485:Protein Binding; D020308:Songbirds; D013045:Species Specificity; D051843:Synucleins; D014020:Tissue Extracts; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D051844:alpha-Synuclein
D009419:Nerve Tissue Proteins; C497605:Snca protein, mouse; D051843:Synucleins; D014020:Tissue Extracts; D014404:Tubulin; D051844:alpha-Synuclein