Proteomic comparison of two fractions derived from the transsynaptic scaffold.
A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF.
Phillips
Greg R
GR
Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA. greg.phillips@mssm.edu
Florens
Laurence
L
Tanaka
Hidekazu
H
Khaing
Zin Z
ZZ
Fidler
Lazar
L
Yates
John R
JR
Colman
David R
DR
D000818:Animals; D002966:Clathrin / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D040901:Proteomics; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry* / Q000648:ultrastructure; D013569:Synapses / Q000737:chemistry* / Q000648:ultrastructure; D013570:Synaptic Membranes / Q000737:chemistry; D013572:Synaptic Vesicles / Q000737:chemistry; D013574:Synaptosomes / Q000737:chemistry
D002966:Clathrin; D009419:Nerve Tissue Proteins