Compartmentation and turnover of the low density lipoprotein receptor in skin fibroblasts.
The low density lipoprotein receptor (LDLR) was immunoprecipitated from [35S]methionine-labeled skin fibroblasts derivatized at 4 or 18 degrees C with an impermeant biotinylating reagent. Separation of derivatized and underivatized receptor from immunoprecipitates by selective binding to streptavidin-agarose allowed assessment of receptor protein cellular compartmentation and rates of intercompartmental transfer. At both 4 and 18 degrees C the amount of LDLR that is derivatized in cells labeled to near steady state saturates after 1-2 h of reaction at, respectively, 47 and 70% of total immunoprecipitable receptor protein. On the basis of temperature titration experiments, protein exposed only to the cell surface reacts at 4 degrees C; raising the temperature of biotinylation to 18 degrees C provides access to an additional pool of receptor protein. Remaining LDLR is derivatized at 37 degrees C. LDLR unreactive at 18 degrees C largely resides in membrane compartment(s) devoid of plasma membrane on the basis of its fractionation on Percoll gradients. While total cellular LDLR and 4 degrees C-derivatized LDLR labeled to steady state turn over in a first order manner (t1/2 = 12-13 h), the specific activity of pulse-labeled, 4 degrees C-accessible protein peaks after 1-2 h of chase and reaches a reduced level by 3 h of chase. These latter results show that the newly synthesized LDLR is transiently enriched at the cell surface prior to achieving equilibrium distribution between the cell surface and intracellular pools.
Hare
J F
JF
Department of Biochemistry, Oregon Health Sciences University, Portland 97201.
D001710:Biotin; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D013696:Temperature
D011973:Receptors, LDL; D001710:Biotin