Two lectin-resistant mutants derived from Madin Darby canine kidney cells, with constitutive alterations in the asparagine-linked carbohydrate moieties, retained the characteristic structural and functional epithelial polarity of the parental cells. A ricin-resistant cell line was unable to incorporate galactose-sialic acid into glycoproteins and, from the pattern of cross-resistance to other lectins, appears to be different from previously described lines resistant to this lectin; the mutation in a concanavalin A-resistant line results, probably, in the production of defective carbohydrate cores of glycoproteins. In spite of glycosylation defects which result in an increased electrophoretic mobility of many cellular glycoproteins, both mutants retained the typical asymmetric structure of the plasma membrane (microvilli on the apical surface, junctional elements on the basolateral surface), functional tight junctions, and unidirectional active transport of electrolytes and water. These results suggest that glycoproteins with terminal galactosesialic acid moieties are not critically involved in the development and maintenance of polarity in epithelial cells. The mutant cells, particularly the ricin-resistant line, exhibited, however, morphological and electrophysiological changes which suggest a quantitative effect of the mutations on intracellular traffic of membranes and tight junction formation. The cell lines described in this paper, the first lectinresistant mutants of epithelial lineage, should prove useful tools for studying the peculiarities of glycosylating pathways in polarized cells.
Most polypeptides destined for secretion are synthesized on polyribosomes bound to the membrane of the endoplasmic reticulum (E.R.), in contrast, cytosolic proteins are made on free ribosomes. When the messenger RNA (mRNA) for a secretory protein is translated in a cell-free protein synthesizing system, the product is usually larger than the mature protein by about 3,000 daltons. Numerous studies have demonstrated that the higher molecular weight of the cell-free translation product can be attributed to an amino terminal extension of about 20-30 amino acids termed the “signal peptide”. This signal peptide is thought to mediate binding of ribosomes bearing the nascent polypeptide chain to the membrane of the endoplasmic reticulum. Upon interaction with the E.R., the polypeptide chain is translocated across the membrane usually resulting in proteolytic removal of the signal peptide and segregation of the “processed” polypeptide into the ER. cisternae. This series of reactions can be followed in vitro by supplementing the cell-free protein synthesizing system with heterologous microsomal membranes which have been stripped of their endogenous ribosomes.
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