Maturation of Lipoprotein Lipase in the Endoplasmic Reticulum: CONCURRENT FORMATION OF FUNCTIONAL DIMERS AND INACTIVE AGGREGATES

Osnat Ben-Zeev,Hui Z Mao,Mark H Doolittle

doi:10.1074/jbc.m108128200

Osnat Ben-Zeev, Hui Z Mao + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m108128200

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Abstract

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.

Highlights

From the Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073 and the Department of Medicine, University of California at Los Angeles, Los Angeles, California 90095
Intracellular lipoprotein lipase (LPL) Is Located Primarily in the endoplasmic reticulum (ER)—In the absence of heparin in the culture medium, LPL is secreted from the cells, a significant amount remains attached to the cell surface through interaction with heparan sulfate proteoglycans
When LPL-depleted Ob17 cells were treated with CCCP, inactive LPL accumulated in the ER

Summary

Introduction

Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. It has been well documented that initial glucose trimming from these nascent glycan chains is a prerequisite for acquisition of LPL catalytic activity (4 – 6) This requirement suggests that, like most glycoproteins, newly synthesized LPL interacts with the netic analysis indicated a concurrent formation of LPL folding chaperones calnexin or calreticulin. Binding to these dimer and aggregate and indicated that the two forms chaperones occurs through the glucose residue remaining on have dissimilar fates. The lack of activity in both triglycerides located within the hydrophobic core of chylomi- cases was attributed to a defect in transport to the Golgi [6]

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