Reichner J S, Whiteheart S W, Hart G W
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1988 Nov 5;263(31):16316-26.
Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.
最近的报告表明，通过高尔基体的大多数分子运输由循环利用的分子组成，而非新合成的分子。为了更详细地评估这种循环途径的重要性，我们使用唾液酸作为共价标记物，研究了小鼠T细胞淋巴瘤EL-4细胞表面糖蛋白的内化和循环利用。通过在4℃下用霍乱弧菌唾液酸酶彻底处理，从活细胞表面去除唾液酸，结果表明其主要来源于糖蛋白（93%），仅有少量来自糖脂（7%）。随着时间的推移，将细胞在37℃下重新培养，并使用针对唾液酸的硫代巴比妥酸测定法的灵敏高压液相色谱法监测细胞表面的再唾液酸化情况。发现唾液酸返回细胞表面取决于从头蛋白质合成，这表明大部分质膜唾液酸糖缀合物不会循环到含有内源性唾液酸转移酶的区室进行寡糖再加工。对人红白血病细胞系K562细胞也得到了相同的结果。使用大鼠肝脏α2-6Galβ1-4GlcNAc唾液酸转移酶以及CMP-[3H]NeuAc作为细胞表面的非渗透性探针，追踪特定糖蛋白的移动。表面唾液酸糖蛋白内化缓慢，这一过程不受环己酰亚胺处理的影响。仅发现这些内化的糖蛋白中有少数返回反式高尔基体区室，随后再循环到细胞表面。综上所述，这些数据表明细胞表面唾液酸的大部分替换是由于糖蛋白的从头合成，并且只有少数糖蛋白通过反式高尔基体区室循环。
