Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates.
The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by beta-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.
Ben-Zeev
Osnat
O
Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, USA.
Mao
Hui Z
HZ
Doolittle
Mark H
MH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D012995:Solubility; D013997:Time Factors; D014162:Transfection
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D004220:Disulfides; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D037281:Calnexin; D000255:Adenosine Triphosphate; D006493:Heparin; D008071:Lipoprotein Lipase; D006026:Glycoside Hydrolases; D019704:Protein Disulfide-Isomerases