XYLOSYLATION IS AN ENDOPLASMIC-RETICULUM TO GOLGI EVENT

The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11097-11104) presented biochemical and subcellular fractionation studies that utilized semi-intact chondrocytes and radiolabeled UDP sugars to overcome obstacles to the direct analysis of xylosylation. The results suggested that xylosylation begins in the endoplasmic reticulum (ER) and continues in the Golgi. The site of xylosylation was not specified further due to the limitations of subcellular fractionation techniques. The studies described in this report were undertaken to localize these modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permeabilization methods utilizing nitrocellulose filter overlays. Biochemical analysis demonstrated the exclusive incorporation of UDP-xylose into the cartilage chondroitin sulfate proteoglycan (aggrecan) core protein and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) into the highly modified proteoglycan monomer. Immunolocalization studies showed the equivalence of cytoplasmic subcompartments in normal and semi-intact chondrocytes at the levels of light and electron microscopy. Once the biochemical and morphological equivalence of intact and semi-intact cells was established, EM autoradiographic studies were pursued using UDP-[H-3]xylose and [S-35]PAPS. Based on both qualitative and quantitative data, silver grains resulting from incorporated sulfate were concentrated in the perinuclear Golgi, while those resulting from incorporated xylose were found at the cis or forming face of the Golgi and in vesicular regions of the peripheral cytoplasm associated with the late ER. These data support the view that xylose addition begins in a late ER compartment and continues in intermediate compartments, perhaps including the cis-Golgi.