Our approach to dissecting the processes of endocytosis, glycoprotein biosynthesis and sorting is through isolation and analysis of mutants. We have previously shown that most CHO cell endocytosis mutants fall into two genetic complementation groups, End1 and End2; both classes of mutants are defective in endosomal, but rot lysosomal, acidification. Having identified a candidate for the End2 protein, Calvin F. Roff has developed a novel preparative three-dimensional gel procedure for purification of this (and other) membrane proteins in quantities sufficient for immunization. To obtain new classes of mutants we devised an isolation procedure for cells defective in lysosomal acidification. Exploiting the quenching of fluorescein at acidic pH we screened for cells exhibiting above normal fluorescence after pulsechase labeling with fluoresceinated dextran (Mr 70,000). One such mutant accumulates dextran in large non-acidic vacuoles; based on functional assays. its endosomal acidification is unimpaired. Susan M. Laurie has continued analysis of LEFIC, a mutant Ltk- cell which is cross-resistant to toxins but has normal endosomal function. The principal defect in LEFIC appears to involve movement of membrane proteins from late Golgi regions to the plasma membrane. Oddly, delivery of membrane proteins in LEFIC is more severely affected than is secretion of soluble proteins. To further characterize mutants defective in early steps in the pathway of N-linked glycosylation, Clara W. Hall has developed an in vitro system for biosynthesis, translocation and elongation of lipid-linked oligosaccharides in intact microsomal vesicles. Conditions for measurement of translocation of lipidlinked Man5GlcNAc2 (the intermediate believed to move from external to luminal faces of the ER) without elongation of the oligosaccharide have beer established.
