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The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [35S] Met with chase periods of 5-24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 microM castanospermine, which is an inhibitor of processing alpha-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR.

Fucose-labelled glycopeptides obtained from the cell surfaces of normal and transformed epithelial cells were compared by co-chromatography on Sephadex G-50. The material obtained from epithelial cells transformed in vitro or from hepatoma cells in culture elutes earlier than the fucose-containing glycopeptides obtained from normal rat epithelial cells. A mutant (TS 223) of a transformed epithelial cell that is temperature-sensitive for maintenance of the transformed phenotype, varies in its Sephadex G-50 profile of cell surface glycopeptides when grown at the permissive (36 degrees C) or the non-permissive temperature (40 degrees C). When grown and labelled at 36 degrees C the gel filtration profile of the glycopeptides resembles that of transformed cells. At 40 degrees C there is an enrichment of later eluting glycopeptides. These differences are more striking in confluent-phase cultures than in log-phase culture. The differences are reversible following upward or downward shifts in growth temperature although there appears to be a lag of at least 6 h before the alteration can be demonstrated by these procedures.

Acid hydrolases are synthesized as precursors that undergo several posttranslational modifications including proteolytic processing to a smaller mature enzyme. The amount of proteolytic processing varies for different acid hydrolases, and many details of the intracellular pathways are not known. The processing of alpha-L-fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse-chase labeling experiments. Only one form of alpha-L-fucosidase, Mr 56,000-57,000, was demonstrated intra- and extracellularly. Under the same conditions, N-acetyl-beta-D-glucosaminidase was shown to be processed with several forms, as previously reported by Hasilik and Neufeld (1980a). To obtain these results, human skin fibroblasts were labeled metabolically with L-[3H]leucine for periods of 20 min to 8 hr with varying periods of chase from 1 to 96 hr with nonradioactive L-leucine. alpha-L-Fucosidase was immunoprecipitated by a polyclonal antibody from material extracted from cells and ammonium sulfate precipitated medium and was examined by polyacrylamide gel electrophoresis under denaturing conditions. N-Acetyl-beta-D-glucosaminidase was examined with similar procedures and served as a control for the methods. Tunicamycin treatment of the cells was used to show that glycosylation did not obscure proteolytic processing because, again, only one form of the intra- and extracellular enzyme was observed, although of smaller size, Mr 52,000-53,000. In addition, separation of the cells into prelysosomal and lysosomal fractions showed only one form of the enzyme. It is concluded that alpha-L-fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.

Glycoproteins from NIH 3T3 transformed by transfection with human tumor DNA had altered glycosylation. That is, the glycoproteins have a decrease in biantennary oligosaccharides with a concomitant increase in tri- and tetraantennary oligosaccharides. After swainsonine treatment, glycosylation of oncogene transfected NIH 3T3 was more similar to the normal counterpart. Thus, this inhibitor of glycoprotein processing is able to reverse at least one phenotypic expression of transformation, the formation of more highly branched oligosaccharides. The mechanism may be by inhibition of a specific glycosyl transferase.