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Journal of Lipid Research, Vol. 45, 1207-1220, July 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Quality control in the apoA-I secretory pathway

Shaila Bhat^1,*, Manal Zabalawi^1,*, Mark C. Willingham^*, Gregory S. Shelness^*, Michael J. Thomas and Mary G. Sorci-Thomas^2,*,

^* Departments of Pathology, Wake Forest University Health Sciences, Winston-Salem, NC
Biochemistry, Wake Forest University Health Sciences, Winston-Salem, NC

² To whom correspondence should be addressed. e-mail: msthomas{at}wfubmc.edu

Manuscript received 3 December 2003, in revised form 11 March 2004, re-revised form 25 March 2004, and in re-re-revised form 30 March 2004.

From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143–186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [³⁵S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that >90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant (6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated 6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation.

These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6.

Supplementary key words apolipoprotein A-I • high density lipoprotein • mutant apolipoprotein A-I • protein secretion • quality control pathway • aggresomes • aggregation • endoplasmic reticulum • Golgi • nuclear envelope • immunofluorescence

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