Hepatic lipase maturation:A partial proteome of interacting factors

Mark H. Doolittle, Osnat Ben-Zeev, Sara Bassilian, Julian P. Whitelegge, Miklós Péterfy, and Howard Wong

Medicine, University of California, Los Angeles and the VA Greater Los Angeles Healthcare System, Los Angels, CA 90073

Corresponding Author: markdool{at}ucla.edu

Tandem-affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in CHO cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, 5 function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase was also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally-misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57 and Glucosidase II, followed by repeated post-translational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding, and may help direct its eventual degradation.

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