XYLOSYLATION IS AN ENDOPLASMIC-RETICULUM TO GOLGI EVENT

The subcellular site of xylosylation, the first carbohydrate modificat ion of the core protein that initiates glycosaminoglycan chain synthes is, was characterized in situ. Methods were developed to combine elect ron microscopic (EM) autoradiography and the radiolabeling of semi-int act chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 110 97-11104) 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 technique s. The studies described in this report were undertaken to localize th ese modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permea bilization methods utilizing nitrocellulose filter overlays. Biochemic al analysis demonstrated the exclusive incorporation of UDP-xylose int o the cartilage chondroitin sulfate proteoglycan (aggrecan) core prote in and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) into the highly mo dified proteoglycan monomer. Immunolocalization studies showed the equ ivalence of cytoplasmic subcompartments in normal and semi-intact chon drocytes at the levels of light and electron microscopy. Once the bioc hemical 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 d ata, silver grains resulting from incorporated sulfate were concentrat ed in the perinuclear Golgi, while those resulting from incorporated x ylose were found at the cis or forming face of the Golgi and in vesicu lar regions of the peripheral cytoplasm associated with the late ER. T hese data support the view that xylose addition begins in a late ER co mpartment and continues in intermediate compartments, perhaps includin g the cis-Golgi.