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Journal of Lipid Research, Vol. 50, 1173-1184, June 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Hepatic lipase maturation: a partial proteome of interacting factors

Mark H. Doolittle^1,*,, Osnat Ben-Zeev^*,, Sara Bassilian^*,, Julian P. Whitelegge^*,, Miklós Péterfy^2,*,,** and Howard Wong^2,*,

^* Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073
Pasarow Mass Spectrometry Laboratory, Neuropsychiatric Institute, Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, CA 90095
^** Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048
² Both authors contributed equally to the work.

This work was supported by the Cedars-Sinai Medical Center, the Department of Veterans Affairs, and Grant HL-24841 from the National Institutes of Health.

Published, JLR Papers in Press, January 8, 2009.

¹ To whom correspondence should be addressed. e-mail: 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 Chinese hamster ovary 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, five 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 were 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 posttranslational 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.

Supplementary key words protein folding • endoplasmic reticulum • chaperones • calnexin • tandem affinity purification • mass spectrometry

Abbreviations: CHO, Chinese hamster ovary; CNX, calnexin; CRT, calreticulin; DSP, dithiobis-(succinimidylpropionate); eEF1A, elongation factor 1-; eLF4A-1, Eukaryotic initiation factor 4A-1; endo H, endoglycosidase H; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum–associated degradation; ERp57, 57 kDa endoplasmic reticulum protein; GII, glucosidase II; Grp78/BiP, 78 kDa glucose-regulated protein; Grp94, 94 kDa glucose-regulated protein; HL, hepatic lipase; Hsp47, 47 kDa heat shock protein; Lmf1, Lipase Maturation Factor 1; MS/MS, tandem mass spectrometry; MSAP, MIR-interacting saposin-like protein; PDI, protein disulfide isomerase; PL, pancreatic lipase; SSR, signal sequence receptor ; TAP, tandem affinity purification; Ub, ubiquitin; UGGT, UDP-glucose:glycoprotein glucosyltransferase 1

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