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, ρ= 1.140 g/cm3; fraction II, ρ= 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. Im‐munoblot analysis for the Na+,K+‐ATPase catalytic subunit, a and α(+), demonstrated that fraction II exhibited predominantly α(+), whose proportion to α 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 (ρ= 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. ALEthylmaleimide (100 μM) completely inhibited the CI‐ uptake in fraction I but partially (∼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.