Identification of a protein related to tubulin in the postsynaptic density.

Abstract

The postsynaptic density is a unique subcellular organelle associated with the synaptic complex and appears as an electron-dense area immediately subjacent to the postsynaptic plasma membrane. The postsynaptic density was isolated from the synaptosomal fraction and the protein constituents were analyzed by polyacrylamide gel electrophoresis. Polypeptides closely related to tubulin were identified as a major component of the postsynaptic density on the basis of molecular weight, subunit structure, and peptide map criteria.Knowledge of the molecular constituents of the synapse is essential in order to understand the underlying mechanisms in the development, maintenance, and plasticity of synaptic connections. At the synapse in the central nervous system, preand postsynaptic membranes are specialized where they are joined. The presynaptic membrane often displays a set of electron-dense projections on its cytoplasmic surface, while on its inner surface the postsynaptic plasma membrane is often distinguished by the presence of an electron-dense structure called the postsynaptic density (PSD). The PSD is not found in all classes of synapses, but in the central nervous system, a prominent PSD is associated with excitatory axodendritic synapses. Neither the functional role nor the exact composition of the PSD is known at present.Identification of the various components of the synapse is dependent on suitable subcellular fractionation techniques. In recent years techniques have been devised for the isolation of synaptic vesicles, synaptic plasma membranes, and most recently, the PSD (1). The PSD fraction can be isolated by treatment of the synaptic plasma membrane fraction with sodium N-lauroyl sarcosinate, followed by density gradient centrifugation. The product of this procedure is a highly purified fraction of PSDs. In the electron microscope isolated PSDs are devoid of their adherent plasma membrane and appear as fibrous and granular matrix which closely resembles that seen in micrographs of intact tissue. Isolated PSDs also retain their distinctive cytochemical properties (1).Isolated PSDs consist primarily of protein with a small amount of carbohydrate and lipid (2). After sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, a single band contains about 40% of the total protein, and therefore this polypeptide(s) probably constitutes the molecular backbone or skeleton of the structure seen in the electron microscope. The apparent molecular weight of this major protein band is 52,000-54,000. On the basis of its molecular weight, which is similar to tubulin, it was suggested that the polypeptide might be tubulin or a very similar protein (2). Tubulin (or a closely related protein) has been identified as a major constituent of both the soluble and insoluble components of the synaptosome (3-5), even though intact microtubules are usually not found at the synapse.In this study we have examined the possibility that tubulin is a major component of the PSD with regard to two criteria: (i) resolution...

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II in PC12 Cells Alters Cell Growth, Morphology, and Nerve Growth Factor-Induced Differentiation

“…The activity of CaMKII was first identified in neuronal membrane fractions (Schulman and Greengard, 1978), and the ␣-subunit of CaMKII was later identified as the major postsynaptic density protein (mPSDp) (Kennedy et al, 1983;Rostas et al, 1983;Kelly et al, 1984;Sahyoun et al, 1985). The mPSDp displays characteristics of a structural protein, such as insolubility in chaotropic agents (Kelly and Cotman, 1976a;Feit et al, 1977), and a propensity to form disulfide bonds with itself and other synaptic proteins (Kelly and Cotman, 1976b). CaMKII is distinct from all known cytoskeletal /structural proteins (Kelly and Cotman, 1976a) and represents 30 -40% of total proteins in PSDs, suggesting that ␣CaMKII could function as a structural /cytoskeletal protein at synapses (Kelly, 1991;Schulman and Hanson, 1993).…”

The postsynaptic density: a possible role in long-lasting effects in the central nervous system.

“…Of the proteins that make up the filamentous structure, actin (2, [10][11][12] and fodrin (13,14) or brain spectrin, an actin-and calmodulin-binding protein, are found in the PSD, with tubulin probably also being a part of the structure (2,10,11,(15)(16)(17)(18). These proteins could account for the tightness of binding of the PSD to the membrane, a tightness that can be inferred from the observations that, in a synaptosomal membrane preparation, only those portions of the presynaptic and postsynaptic membranes at the synapse are connected to each other (3), as if bound together through the proteins of the PSD linking it to the postsynaptic membrane, and probably via the synaptic cleft, linking the pre-and postsynaptic membranes.…”

Alterations in Protein and Glycoprotein Composition of Rat Embryo Fibroblasts Transformed by Herpes Simplex Virus Type 2

“…It is possible that another 56,000-dalton HSV-trans formation-sensitive protein corresponds to the tubulin subunit which was demonstrated to be an integral com ponent of both the plasma membrane and the intracytoplasmic membrane system [30].…”

“…This included superficially labeled sialopeptides of high 130,000, 100,000 and 76,000 daltons) and low (30,000 and 15,000 daltons) molecular weights.…”