ISOLATION OF 3 CLASSES OF CONDITIONAL-LETHAL CHINESE-HAMSTER OVARY CELL MUTANTS WITH TEMPERATURE-DEPENDENT DEFECTS IN LOW-DENSITY-LIPOPROTEIN RECEPTOR STABILITY AND INTRACELLULAR MEMBRANE-TRANSPORT

Fluorescence-activated cell sorting was used to isolate 19 independent , temperature-sensitive, low density lipoprotein (LDL) receptor-defici ent Chinese hamster ovary cell mutants that define three recessive com plementation groups, IdlE, ldlF, ldlG. LDL receptor activity, essentia lly normal at the permissive temperature (34 degrees C), was virtually abolished in the mutants after incubation for 8-12 h at the nonpermis sive temperature (39-40.5 degrees C). The mutants died after incubatio n for >24 h at 39.5 degrees C. These mutants exhibited two striking an d unexpected abnormalities that suggest that they define three genes i mportant for general vesicular membrane traffic. First, LDL receptors were degraded abnormally rapidly at the nonpermissive temperature (chl oroquine inhibited this degradation in ldlE and ldlG, but not in ldlF) . In IdlE cells, the rapid degradation did not require efficient recep tor clustering into coated pits and was not observed for all cell surf ace proteins. This selective degradation may be due to endocytic misso rting. Second, the mutants exhibited temperature-sensitive defects in the posttranslational processing and intracellular transport of many m embrane-associated and secreted proteins, including the LDL, mannose 6 -phosphate/insulin-like growth factor II, and scavenger receptors, the vesicular stomatitis virus G protein and decay accelerating factor. A lthough the initial synthesis, folding, and processing of precursor fo rms of these proteins in the endoplasmic reticulum were apparently nor mal at the nonpermissive temperature, there was either a delay or a bl ock in oligosaccharide processing associated with endoplasmic reticulu m to medial Golgi transport at the nonpermissive temperature. This was accompanied by a dramatic inhibition of total soluble protein secreti on. The posttranslational processing defects, the instability of cell surface LDL receptors, and the defective protein secretion exhibited b y these mutants suggest that the ldlE-G gene products regulate or part icipate in reactions that are vital for a variety of secretory and end ocytic membrane transport processes. This suggestion is strongly suppo rted by our recent observation that a cDNA encoding a component of the coatomer, epsilon-COP, corrects the mutant phenotypes of ldlF cells ( Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-12 24). Thus, these mutant cells should prove useful for further genetic and biochemical analysis of the mechanisms underlying intracellular me mbrane traffic.