Two Types of Clathrin‐Coated Vesicles Isolated from Rat Brain: Analysis of Biochemical Properties and Cellular Origin

Abstract

Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.

A depolarization‐stimulated, bafilomycin‐inhibitable H+ pump in hippocampal astrocytes

“…Recent studies have indicated that astrocytes contain such clathrin-coated vesicles (Uriu et al, 1991). There are three general categories of ion-translocating ATPases.…”

“…The v-type H+-ATPase was initially isolated in clathrin-coated vesicles from bovine brain (Stone et al, 1983). Recent studies have indicated that astrocytes contain such clathrin-coated vesicles (Uriu et al, 1991). There are three general categories of ion-translocating ATPases.…”

Regulation of neuronal function by protein trafficking: a role for the endosomal pathway

“…Two populations of primary endocytic vesicles have been described that are likely to correspond to the direct and indirect pathways for synaptic vesicle biogenesis. Coated vesicle preparations from brain have been shown to contain at least two distinct types of coated vesicles (Uriu et al 1991; Thoidis et al 1998, 1999). One population contains only synaptic vesicle proteins, while the second population contains synaptic vesicle proteins along with other potential nerve terminal proteins and can be labelled with antibodies to the extracellular domain of included proteins (Thoidis et al 1998, 1999; Provoda et al 2000).…”

Ultrastructural Pathology of Plasma and Endoplasmic Reticulum Membranes of Nerve and Glial Cells: A Review

“…Endocytosis is a fundamental mechanism by which neurons control intercellular signal, nutrient uptake, and synaptic transmission [70]. Clathrin-coated vesicles of different sizes have been isolated from rat brain, and related with the content of Na+, K (+)-ATPase, as a mechanism of Cl-uptake [71], as pathways for endocytosis of neural cell adhesion molecules (NCAM) [72], and for receptor-mediated vesicular transport [73], such as internalization of metabotropic glutamate receptors [74].…”