Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes

Treatment with NH4Cl of mouse thymocytes renders their plasma membrane permeable to sugar nucleotides both inwards and outwards. Using this model, we studied the entry and utilization of CMP-NeuAc, GDP-Fuc and UDP-Gal into intracellular vesicles in situ. It is shown that CMP-NeuAc and GDP-Fuc enter the vesicles in a manner indicating a carrier-mediated transport (substrate saturation curve, inhibition by substrate analogues, temperature dependence) and are entrapped in their uncleaved form. This leads to the formation of an intralumenal pool of these precursors which can be further utilized by the sialyltransferases and fucosyltransferases. The occurrence of an endogenous pool of CMP-NeuAc and GDP-Fuc is demonstrated by the fact that, when the vesicles are disrupted by detergent, the release of the endogenous sugar nucleotides causes an isotopic dilution of the labelled precursors added to measure the glycosyltransferase activities. In contrast, no accumulation of UDP-Gal has been detected, suggesting that transport and transfer reaction are simultaneous events. However, experiments with UDP 2′,3′-dialdehyde indicate that UDP-Gal is not transported through the membrane by galactosyltransferase action but by a distinct carrier molecule.