Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.
A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.
Malmström
K
K
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Krieger
M
M
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D006224:Cricetinae; D003470:Culture Media; D004705:Endocytosis / Q000528:radiation effects; D007700:Kinetics; D037102:Lectins / Q000633:toxicity; D008077:Lipoproteins, LDL / Q000378:metabolism*; D016296:Mutagenesis; D011973:Receptors, LDL / Q000378:metabolism*; D013696:Temperature
D003470:Culture Media; D037102:Lectins; D008077:Lipoproteins, LDL; D011973:Receptors, LDL