Identification of proteins in the postsynaptic density fraction by mass spectrometry

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in st ructures with the morphology of PSDs. In this study, we have completed the identification of most major proteins in the PSD fraction with the use of a n analytical method based on mass spectrometry coupled with searching of th e protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 pro teins not previously known to be constituents of the PSD fraction and 24 th at had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mamma lian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast. Both myosin-Va and cdc10 are threefold to fivefold enri ched in the PSD fraction over brain homogenates. Immunocytochemical localiz ation of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons. Cdc10 has a punctate distribution in cell bo dies and dendrites, with some of the puncta colocalizing with PSD-95. The r esults support a role for myosin-Va in transport of materials into spines a nd for septins in the formation or maintenance of spines.