Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions.
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 [(35)S]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 (Delta 6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated Delta 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.
Bhat
Shaila
S
Department of Pathology, Wake Forest University Health Sciences, Winston-Salem, NC, USA.
Zabalawi
Manal
M
Willingham
Mark C
MC
Shelness
Gregory S
GS
Thomas
Michael J
MJ
Sorci-Thomas
Mary G
MG
D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021381:Protein Transport; D017384:Sequence Deletion; D014162:Transfection
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010743:Phospholipids