Prechylomicron transport vesicle: isolation and partial characterization

Abstract

The intestine is able to regulate its output rate of chylomicrons, the major intestinal triacylglycerol (TG) transport vehicle. We have proposed that a vesicle, transporting the developing chylomicron from the endoplasmic reticulum (ER) to the Golgi, is the rate-limiting step in the process of TG transit through the enterocyte [Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G18-G30, 1997]. We wished to isolate and characterize this vesicle. The apical portion of rat intestinal cells were avulsed, and the mucosa was stirred in buffer. The supernatant was centrifuged in two different sucrose gradients, and the top 2.5 ml of the last gradient were collected and concentrated. Electron microscopy showed a 200-nm vesicle. The vesicle contained immunoidentifiable apolipoprotein (apo) B48 and apo A-IV but very little apo A-I, although apo A-I was present in the ER and Golgi. [3H]TG-loaded vesicles delivered [3H]TG to the Golgi but not the ER. Marker enzyme assays also indicate that the isolated fraction is different from the ER and Golgi fractions. We conclude that we have isolated a vesicle that is post-ER but pre-Golgi that vectorially transports TG to the Golgi.